BRPI0619133B1 - CHIMERIC NEWCASTLE DISEASE VIRUS, IMMUNOGENIC COMPOSITION, METHOD FOR PRODUCING AN IMMUNOGENIC FORMULATION, AND, USE OF A CHIMERIC VIRUS - Google Patents

Abstract

CHIMERIC INFLUENZA VIRUS, RECOMBINANT DNA MOLECULE, METHOD FOR PRODUCING AN IMMUNOGENIC FORMULATION, ATTENUATED CHIMERIC INFLUENZA VIRUS, CHIMERIC NDV, AND, USE OF A CHIMERIC VIRUS The present invention provides chimeric negative-strand RNA viruses that allow an individual, for example a bird, being immunized against two infectious agents using a single chimeric virus of the invention. Particularly, the present invention provides chimeric influenza viruses engineered to express and incorporate into their virions a fusion protein comprising an ectodomain of an infectious agent protein and the transmembrane and cytoplasmic domain of an influenza virus protein. These chimeric viruses induce an immune response against influenza viruses and the infectious agent. The present invention also provides chimeric Newcastle disease virus (NDV) engineered to express and incorporate into its virions a fusion protein comprising the ectodomain of a protein from an infectious agent and the transmembrane and cytoplasmic domain of an NDV protein. These chimeric viruses induce an immune response against NDV and the infectious agent.

Description

1. FIELD OF INVENTION

[0001] The present invention provides chimeric negative-strand RNA viruses that allow an individual, for example, a bird, to be immunized against two infectious agents using a single chimeric virus of the invention. In particular, the present invention provides chimeric influenza viruses engineered to express and incorporate into their virions a fusion protein comprising an ectodomain of a protein from an infectious agent and the transmembrane and cytoplasmic domain of an influenza virus protein. These chimeric viruses induce an immune response against influenza viruses and the infectious agent. The present invention further provides chimeric NewCastle disease virus (NDV) engineered to express and incorporate into its virions a fusion protein comprising the ectodomain of a protein from an infectious agent and the transmembrane and cytoplasmic domain of an NDV protein. These chimeric viruses induce an immune response against NDV and the infectious agent.

2. BACKGROUND OF THE INVENTION

[0002] Various DNA viruses have been genetically engineered to direct the expression of heterologous proteins in host cell systems (e.g., vaccinia virus, baculovirus, etc.). Recently, similar advances are made with positive-strand RNA viruses (e.g., poliovirus). The expression products of these constructs, i.e., the heterologous gene product or the chimeric virus expressing the heterologous gene product, are considered potentially usable in vaccine formulations (both subunit and whole virus vaccines). A drawback to using viruses, such as vaccinia, to construct recombinant or chimeric viruses for use in vaccines is the lack of variation in their key epitopes. This lack of variability in viral strains places strict limitations on the repeated use of chimeric vaccinia, in which multiple vaccinations will generate host resistance to the strain so that the inoculated virus cannot

[0003] In contrast, negative-strand RNA viruses are attractive candidates for constructing chimeric viruses for use in vaccines. Negative-strand RNA viruses, e.g., influenza, are desirable because their wide genetic variability allows the construction of a vast repertoire of vaccine formulations that stimulate immunity without the risk of developing tolerance.

2.1 Negative-strand RNA viruses

[0004] The families of viruses containing enveloped single-stranded RNA of the negative sense genome are classified into strains having non-segmented genomes (Paramyxoviridae, Rhabdoviridae) or those having segmented genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). The families Paramyxoviridae and Orthomyxoviridae are described in detail below and used in the examples here. The family Paramyxoviridae contains the viruses NewCastle disease virus (NDV), parainfluenza virus, Sendai virus, simian virus 5, and mumps virus. The Orthomyxoviridae family contains influenza viruses, types A, B, and C viruses, as well as Thogoto and Dhori viruses and infectious salmon anemia viruses.

2.1.1 Influenza virus

[0005] Influenza virions comprise an internal ribonucleoprotein core (a helical nucleocapsid) containing the single-stranded RNA genome, and a lipoprotein envelope lined internally by a matrix protein (M1). The segmented genome of the influenza A virus consists of eight molecules (seven for influenza C) of linear, negative-polarity, single-stranded RNAs encoding ten polypeptides, including: the RNA-dependent RNA polymerase proteins (PB2, PB1, and PA ) and nucleoprotein (NP) that form the nucleocapsid; the matrix membrane proteins (M1, M2); two surface glycoproteins that protrude from the lipid-containing envelope: hemagglutinin (HA) and neuraminidase (NA); the non-structural protein (NS1) and nuclear export protein (NEP). Transcription and replication of the genome take place in the nucleus and assembly occurs via budding on the plasma membrane. Viruses can regroup during mixed infections.

[0006] The influenza virus adsorbs into cells via HA binding activity to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. After endocytosis of the virion, a conformational change in the HA molecule occurs in the cellular endosome that facilitates membrane fusion, thus triggering the non-formation of the coat. The nucleocapsid migrates to the nucleus where the viral mRNA is transcribed. Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the 5' terminus of cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the addition of poly(A) areas. The viral RNA transcripts then migrate to the cell membrane and associate with recently described viral transmembrane proteins. NA then cleaves sialy residues from the carbohydrate moieties of membrane-bound glycoproteins resulting in the encapsulation and cellular release of the progeny virus. Of the eight viral RNA molecules thus produced, six are monocistronic messages that are translated directly into proteins representing HA, NA, NP and the viral polymerase proteins PB2, PB1 and PA. The other two transcripts are spliced, each giving two mRNAs that are translated into different reading frames to produce M1, M2, NS1, and NEP. In other words, the eight viral RNA segments encode ten proteins: nine structural and one non-structural. A summary of influenza virus genes and their protein products is shown in Table 1 below. Table 1: Influenza virus genome RNA segments and coding designations

[0007] The pathogenicity of influenza viruses is modulated by multiple virus and host factors. Among the host factors that combat viral infections, the type I interferon (IFNa/ß) system represents a powerful innate antivirus defense mechanism that was established relatively early in the evolution of eukaryotic organisms (Garcia-Sastre, 2002, Microbes Infect. 4 :647-55). The antivirus IFNa/ß system involves three main steps: (i) detection of viral infection and IFNa/ß secretion, (ii) binding of IFNa/ß to its receptors and transcriptional induction of genes stimulated by IFNa/ß, and (iii ) synthesis of antiviral enzymes and proteins. Most viruses, however, acquire specific genetic information encoding IFNa/ß antagonist molecules, which effectively block one or more steps of the antiviral IFNa/ß system. Influenza A viruses express a non-structural protein from infected cells, the NS1 protein (described in detail, below), which opposes the cellular IFNa/ß response (Garcia-Sastre et al., 1998, Virology 252: 324 -30).

2.1.1.1 Highly pathogenic avian influenza

[0008] In recent years, outbreaks of highly pathogenic avian influenza (HPAI) have been reported in Asia and Europe (Kawaoka et al., 2005, Natl. Rev. Microbiol, 3: 591-600; Koopmans et al., 2004 , Lancet 363: 587-593). Outbreaks involving influenza A, subtype H5N1 or H7N7 viruses have resulted in lethal infections in poultry, and the death of a limited number of human cases (Tweed et al., 2004, Emerg. Infec. Dis. 10: 2196-2199). The current H5N1 viruses have been circulating among poultry in China in recent years (Chen et al., 2005, Nature 436: 191-192), and while migratory birds are being considered to be the primary reservoir of these viruses, it is believed that transmission from infected domestic birds back to migratory birds has contributed to their increased geographic distribution. Currently, the H5N1 virus has emerged in Asia, spreading through Europe and Africa (Enserink, 2006, Science, 311-932). Refusal to wholesale poultry has been shown to be a successful strategy in eradicating H5N1 outbreaks in Hong Kong in 1997 and the Netherlands in 2003 (Lipatov et al., 2004, J. Virol. 78: 8951-8959 ). Since human victims of recent H5N1 outbreaks have had close contact with infected poultry, it follows that prevention of interspecies transmission of avian influenza viruses (AIV) can be accomplished by eradicating AIV in poultry through culling. However, for economic and practical reasons, destruction of infected poultry alone is no longer considered the method of choice in controlling this disease. Furthermore, for ethical and ecological reasons, the rejection of edible wild migratory birds is considered an unacceptable practice. Recently, the OIE (World Organization for Animal Health) and FAO (Food and Agriculture Organization of the United Nations) recommended that vaccination of poultry should be considered for AIV control. Furthermore, it was reported that vaccinating chickens with inactivated H5 vaccine was successful in interrupting transmission of the virus in a field study (Ellis et al., 2004, Avian Pathol. 33: 405-412. Recently, China has accepted vaccination as a component of its AIV control program.

[0009] The possibility that the highly pathogenic H5N1 strain could become transmissible between humans is referenced in terms of a global pandemic, with the WHO reluctant to estimate global mortality should the H5N1 virus recombine in human form. Thus, the need for a method of controlling H5N1 infection in agricultural cargoes, from which most transmissions to humans are believed to have arisen, is evident.

2.1.2 Newcastle disease virus

[0010] Newcastle disease virus is an enveloped virus containing a linear, single-stranded, non-segmented, negative-sense RNA genome. Genomic RNA contains genes in the order of 3'-N-P-M-F-HN-L, described in detail below. Genomic RNA also contains a leader sequence at the 3' end.

[0011] The structural elements of the virion include the viral envelope, which is a lipid bilayer derived from the cell's plasma membrane. The glycoprotein, hemagglutinin-neuraminidase (HN) projects from the envelope providing both hemagglutinin (e.g., fusogenic/receptor binding) and neuraminidase activities. The fusion glycoprotein (F), which also interacts with the viral membrane, is first produced as an inactive precursor, then cleaved post-translationally to produce two disulfide-linked polypeptides. The active F protein is involved in the penetration of NDV into host cells by facilitating the fusion of the viral envelope with the host cell plasma membrane. The matrix protein (M) is involved in viral assembly, and interacts with both the viral membrane and nucleocapsid proteins.

[0012] The main protein subunit of the nucleocapsid is the nucleocapsid protein (N) which confers helical symmetry on the capsid. In association with the nucleocapsid are the P and L proteins. The phosphoprotein (P), which is subject to phosphorylation, is considered to play a regulatory role in transcription, and may also be involved in methylation, phosphorylation, and polyadenylation. The L gene, which encodes an RNA-dependent RNA polymerase, is required for the synthesis of viral RNA along with the P protein. The L protein, which absorbs almost half of the coding capacity of the viral genome, is the largest of the viral proteins, and plays an important role in both transcription and replication.

[0013] Replication of all negative-strand RNA viruses, including NDV, is complicated by the absence of the cellular machinery required to replicate RNA. Furthermore, the negative-strand genome cannot be directly translated into protein, but must first be transcribed into a positive-strand copy (mRNA). Thus, upon entry into a host cell, the virus cannot synthesize the required RNA-dependent RNA polymerase. The L, P and N proteins must enter the cell along with the genome during infection.

[0014] It was hypothesized that most or all of the viral proteins that transcribe NDV mRNA also carry out its replication. The mechanism that regulates alternative uses (i.e., transcription or replication) of the same complement of proteins has not been clearly identified but appears to involve an abundance of free forms of one or more of the nucleocapsid proteins, in particular, N. Directly after Virus penetration, transcription is initiated by the L protein using the negative-sense RNA in the nucleocapsid as a template. Viral RNA synthesis is regulated so that it produces monocistronic mRNAs during transcription.

[0015] After transcription, replication of the viral genome is the second essential event in infection by negative-strand RNA viruses. As with other negative-strand RNA viruses, viral genome replication in Newcastle disease virus (NDV) is mediated by virus-specified proteins. The first products of replicative RNA synthesis are complementary (i.e., positive polarity) copies of NDV genome RNA (cRNA). These plus-strand copies (antigenomes) differ from plus-strand mRNA transcripts in the structure of their termini. Unlike mRNA transcripts, anti-genomic cRNAs are not terminated and methylated at the 5' ends, and are not truncated and polyadenylated at the 3' ends. cRNAs are co-terminal with their negative-strand templates and contain all of the genetic information in each genomic RNA segment in complementary form. cRNAs serve as templates for the synthesis of NDV negative-strand viral genomes (vRNAs).

[0016] Both NDV negative strand genomes (vRNAs) and antigenomes (cRNAs) are encapsidated by nucleocapsid proteins; the only non-encapsidated RNA species are the virus's mRNAs. For NDV, the cytoplasm is the site of viral RNA replication, just as it is the site for transcription. The assembly of viral components appears to take place in the plasma membrane of the host cell and mature virus is released by budding.

2.2 Immunogenic formulations

[0017] Recombinant DNA technology and “reverse genetics” engineering techniques provide a unique approach to the production of recombinant viruses for use in immunogenic formulations. In particular, the present invention provides a method for engineering a negative-strand RNA virus so that it expresses, or demonstrates, not only native viral antigens, but also any antigen that can be engineered to incorporate into the viral protein coat. Of particular interest are antigens derived from infectious organisms other than influenza. In this way, a single virus can be engineered as an immunogenic compound usable to elicit, activate or induce an immune response that would provide protection against at least two pathogens. This chimeric virus can be further engineered when necessary to modify its virulence, that is, so that it can be attenuated or further attenuated. Attenuated influenza viruses are beneficial because they are immunogenic and capable of replication, but not pathogenic.

[0018] Live vaccines are thought to induce enhanced cross-reactive cell-mediated cytotoxicity as well as a humoral antibody response, providing better protection than inactivated vaccines (Gorse and Belshe, 1990, J. Clin. Microbiol. 28: 2539 - 2550, and Gorse et al, 1995, J. Infect. Dis. 172: 1-10). Second, protective immunity to viral diseases will likely involve mucosal IgA response that is not seen with traditional intramuscularly administered vaccines. (Nelson et al, 1998, Vaccine, 16: 1306-1313). Finally, live vaccines also have the advantage of intranasal administration which avoids the swelling and muscle soreness occasionally associated with intramuscular administration of vaccines with inactivated adjuvants. These live vaccines have been reported to induce not only humoral responses against homotypic influenza viruses but also cross-reactive cell-mediated cytotoxicity. Thus, the invention offers the potential for the development of new and more effective immune formulations, for example, vaccine formulations, for the diagnosis, prevention, control or treatment of both viral and non-viral pathogens.

3. SUMMARY OF THE INVENTION

[0019] The present invention provides negative-strand RNA chimeric viruses engineered to express fusion proteins that incorporate into the virion, methods for producing such chimeric viruses, and the use of such viruses, for example as immunogens, in immunogenic formulations, or in assays. in vitro. The chimeric viruses of the invention are characterized by displaying, on the surface of the virion, not only antigens associated with the virus but also the fusion protein.

[0020] The present invention provides chimeric influenza viruses and chimeric NDVs that allow an individual, for example, avian or human, to be immunized against two infectious agents by administering a chimeric influenza virus or a chimeric NDV. In one aspect, the use of a single virus to induce an immune response reduces the frequency of administration of an immunizing formulation. In another aspect, using a single virus to induce an immune response reduces the cost of immunizing individuals. The lower cost of immunizing individuals increases the likelihood that more individuals will be able to afford to be immunized and thus reduces healthcare costs associated with treating individuals suffering from an infection.

[0021] The invention also relates to the use of a chimeric virus of the invention in compositions (e.g., immunogenic formulations) for humans or animals. In particular, the chimeric viruses of the invention can be used as vaccines against a broad range of viruses and/or antigens. Because the chimeric virus is engineered to express foreign epitopes on the virion, compositions (e.g., vaccine formulations) comprising a chimeric virus of the invention can be engineered for immunization against multiple strain variants, different viruses, or against completely different infectious agents or antigens. disease (e.g., bacteria, parasites, fungi, or tumor-specific antigens). Many methods can be used to introduce live attenuated virus formulations into a human or animal subject to induce an appropriate immune or cytokine response. These include, but are not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.

[0022] The chimeric viruses of the invention enable an individual (e.g., bird) to be immunized for two infectious diseases by administering the chimeric viruses. In a specific embodiment, the chimeric viruses of the invention enable birds to be immunized for avian influenza viruses and Newcastle disease viruses by administering a chimeric virus of the invention. Birds can be

[0023] The present invention is based, in part, upon Applicants' discovery that an effective immune response to two infectious agents can be accomplished by engineering an influenza virus to express and incorporate within its virions a fusion protein comprising the domains cytoplasmic and transmembrane domain of at least one essential glycoprotein of the virus and the ectodomain of a protein of a second infectious agent, wherein the fusion protein functionally replaces the essential glycoprotein. In one aspect, incorporation of the fusion protein into the virion results in an enhanced immune response to the ectodomain of the second infectious agent. Engineering the cytoplasmic and transmembrane domains of an essential virus glycoprotein into the fusion protein allows the fusion protein to incorporate into the virion. In a particular embodiment, the essential glycoprotein is one or both of the influenza virus NA and/or HA protein. In another embodiment, the essential glycoprotein is one or both of the NDV F or HN protein. Functional replacement of at least one essential glycoprotein of the virus eliminates concerns about limiting the size of the virus genome (e.g., the influenza virus genome). In some embodiments, functional replacement of at least one essential glycoprotein of the virus with the fusion protein attenuates viral replication in individuals.

[0024] The present invention provides a chimeric avian influenza virus, comprising a fusion protein, having (i) an ectodomain of a protective antigen from an infectious agent, different from the influenza virus, fused to (ii) a transmembrane domain and cytoplasmic glycoprotein encoded by an essential gene of an influenza virus, wherein the fusion protein is incorporated into an avian influenza virus, wherein the function of the essential gene is supplied by the fusion protein or by the glycoprotein native to the avian influenza virus. In some embodiments, the essential gene of an influenza virus is a hemagglutinin (HA) gene. In other embodiments, the essential gene of a

[0025] The present invention provides a chimeric avian influenza virus, comprising a fusion protein, having (i) an ectodomain of an NDV HN protein fused to (ii) a transmembrane and cytoplasmic domain of an NDV NA protein. influenza, wherein the fusion protein is incorporated into an avian influenza virus, wherein the function of the NA protein is supplied by a fusion protein or by the glycoprotein native to the avian influenza virus. In some embodiments, the chimeric avian influenza virus is attenuated. According to these embodiments, the chimeric avian influenza virus can be attenuated by mutations in the NS1 gene. According to the invention, any type, subtype or strain of avian influenza virus can be used.

[0026] The present invention provides a chimeric avian influenza virus, comprising an enveloped influenza virus NA segment encoding a neuraminidase fusion protein, wherein the NA open reading frame is modified such that the nucleotides encoding the ectodomain of NA are replaced by nucleotides encoding an ectodomain of a neuraminidase antigen from an infectious agent other than influenza that is anchored by the N-terminus, so that the neuraminidase fusion protein is expressed and incorporated into the chimeric avian influenza virus.

[0027] The present invention provides a chimeric avian influenza virus, comprising an enveloped influenza virus HA segment encoding a hemagglutinin fusion protein, wherein the HA open reading frame is modified so that the nucleotides encoding the ectodomain of HA are replaced by nucleotides encoding an ectodomain of a fusogenic/binding receptor antigen from an infectious agent other than influenza virus that is anchored by the C-terminus, so that the hemagglutinin fusion protein is expressed and incorporated into the virus. chimeric avian influenza.

[0028] The present invention provides a chimeric avian influenza virus, comprising a bicistronic enveloped influenza virus HA segment, comprising: (a) a first open reading frame encoding an avian influenza virus hemagglutinin protein, and (b) a second open reading frame encoding a hemagglutinin fusion protein, in which the nucleotides encoding the hemagglutinin ectodomain are replaced by nucleotides encoding an ectodomain of a protective antigen of an infectious agent other than influenza virus, or encoding a disease antigen that is anchored by the C-terminus such that both the influenza virus hemagglutinin and the fusion protein are expressed and incorporated into the chimeric avian influenza virus. In some embodiments, the first open reading frame of the HA segment of the avian chimeric virus is modified to remove the hemagglutinin polybasic cleavage site.

[0029] The present invention provides a chimeric avian influenza virus, comprising a bicistronic enveloped influenza virus NA segment, comprising: (a) a first open reading frame encoding an avian influenza virus neuraminidase protein, and (b) a second open reading frame encoding a neuraminidase fusion protein, in which the nucleotides encoding the neuraminidase ectodomain are replaced by nucleotides encoding an ectodomain of a protective antigen of an infectious agent, other than influenza virus, or encoding a disease antigen that is anchored by the N-terminus, so that both the influenza neuraminidase virus neuraminidase and the fusion protein are expressed and incorporated into the chimeric avian influenza virus. In some embodiments, the chimeric avian influenza virus comprises an HA segment having an open reading frame modified to remove the hemagglutinin polybasic cleavage site.

[0030] The present invention provides a chimeric avian influenza virus, comprising an enveloped influenza virus NA segment encoding a neuraminidase fusion protein, wherein the NA open reading frame is modified such that the nucleotides encoding the ectodomain of NA are

[0031] In some embodiments, a chimeric avian influenza virus of the invention comprises an enveloped NS1 gene segment encoding a modified NS1 protein that reduces the cellular interferon antagonist activity of the virus. Non-limiting examples of mutations in the NS1 gene that result in a modified NS1 protein are provided in Section 5.1.2, infra.

[0032] The present invention provides recombinant nucleic acid molecules (e.g., recombinant DNA molecules) encoding the NA segment of the chimeric avian influenza viruses of the invention. The present invention further provides recombinant nucleic acid molecules (e.g., recombinant DNA molecules) encoding the HA segment of the chimeric avian influenza virus of the invention. The present invention further provides recombinant nucleic acid molecules (e.g., recombinant RNA molecules) encoding the NA segment or the HA segment of the chimeric avian influenza virus of the invention.

[0033] The present invention provides methods for propagating a chimeric avian influenza virus of the invention, comprising cultivating the chimeric avian influenza virus in an embryonated egg or a cell line that is susceptible to infection by avian influenza viruses. The present invention further provides methods for producing an immunogenic formulation, the method comprising: (a) propagating a chimeric avian influenza virus of the invention in an embryonated egg or a cell line that is susceptible to infection by avian influenza viruses; and (b) collecting the progeny virus, where the virus is grown in sufficient quantities and under sufficient conditions so that the virus is free from contamination, so that the progeny virus is suitable for use in immunogenic formulations, e.g. vaccine formulations.

[0034] The present invention provides an attenuated chimeric influenza virus, comprising a fusion protein, having (i) an ectodomain of a protective antigen from an infectious agent, different from the influenza virus fused to (ii) a cytoplasmic and transmembrane and a glycoprotein encoded by an essential gene of an influenza virus, wherein the fusion protein is incorporated into an attenuated influenza virus, wherein the function of the essential gene is supplied by the fusion protein or by the glycoprotein native to the attenuated influenza virus. In some embodiments, the essential gene of an influenza virus is a hemagglutinin (HA) gene. In other embodiments, the essential gene of an influenza virus is a neuraminidase (NA) gene. The attenuated chimeric influenza virus can be any type, subtype, or strain of influenza virus. For example, the attenuated chimeric influenza virus may be an influenza A virus, an influenza B virus, or an influenza C virus.

[0035] The present invention provides an attenuated chimeric influenza virus comprising an enveloped influenza virus NA segment encoding a neuraminidase fusion protein, wherein the NA open reading frame is modified such that the nucleotides encoding the ectodomain of NA are replaced by nucleotides encoding an ectodomain of a neuraminidase antigen from an infectious agent other than influenza that is anchored by the N-terminus, so that the neuraminidase fusion protein is expressed and incorporated into the attenuated chimeric influenza virus. In some embodiments, the attenuated chimeric influenza virus of the invention comprises an HA segment having an open reading frame modified to remove the hemagglutinin polybasic cleavage site.

[0036] The present invention provides an attenuated chimeric influenza virus, comprising an influenza virus HA segment

[0037] enveloped encoding a hemagglutinin fusion protein, in which the HA open reading frame is modified such that nucleotides encoding the ectodomain of HA are replaced by nucleotides encoding an ectodomain of a hemagglutinin antigen from a different infectious agent of influenza that is anchored by the C-terminus, so that the hemagglutinin fusion protein is expressed and incorporated into the attenuated chimeric influenza virus.

[0038] The present invention provides an attenuated chimeric influenza virus, comprising a bicistronic enveloped influenza virus HA segment, comprising: (a) a first open reading frame encoding influenza hemagglutinin protein, and (b) a second open reading frame encoding a hemagglutinin fusion protein, in which nucleotides encoding the hemagglutinin ectodomain are replaced by nucleotides encoding an ectodomain of a protective antigen of an infectious agent, other than influenza, or encoding a disease antigen which is anchored by the C-terminus, so that both the influenza hemagglutinin and the fusion protein are expressed and incorporated into the attenuated chimeric influenza virus. In some embodiments, the first open reading frame of the HA segment of the attenuated chimeric influenza virus is modified to remove the hemagglutinin polybasic cleavage site.

[0039] The present invention provides an attenuated chimeric influenza virus, comprising a bicistronic enveloped influenza virus NA segment, comprising: (a) a first open reading frame encoding an influenza neuraminidase protein, and (b) a second open reading frame encoding a neuraminidase fusion protein, in which nucleotides encoding the neuraminidase ectodomain are replaced by nucleotides encoding an ectodomain of a protective antigen of an infectious agent, other than influenza, or encoding a disease antigen which is anchored by the N-terminus, so that both the influenza neuraminidase and the fusion protein are expressed and incorporated into the attenuated chimeric influenza virus. In some embodiments, the attenuated chimeric influenza virus of the invention comprises an HA segment having an open reading frame modified to remove the hemagglutinin polybasic cleavage site.

[0040] In some embodiments, the attenuated chimeric influenza virus of the invention comprises an enveloped NS1 gene segment encoding a modified NS1 protein that reduces the cellular interferon antagonist activity of the virus.

[0041] The present invention provides recombinant nucleic acid molecules (e.g., recombinant DNA molecules) encoding the NA segment of the attenuated chimeric influenza viruses of the invention. The present invention further provides recombinant nucleic acid molecules (e.g., recombinant DNA molecules) encoding the HA segment of the attenuated chimeric influenza viruses of the invention. The present invention further provides recombinant nucleic acid molecules (e.g., recombinant RNA molecules) encoding the NA segment or HA segment of the attenuated chimeric influenza viruses of the invention.

[0042] The present invention provides methods for propagating the attenuated chimeric influenza virus of the invention, comprising culturing the attenuated chimeric influenza virus in an embryonated egg or a cell line that is susceptible to influenza virus infection. The present invention further provides methods for producing an immunogenic formulation, the method comprising: (a) propagating an attenuated chimeric influenza virus of the invention in an embryonated egg or a cell line that is susceptible to infection by attenuated influenza viruses; and (b) collecting the progeny virus, wherein the virus is grown in sufficient quantities and under sufficient conditions that the virus is free from contamination, so that the progeny virus is suitable for use in immunogenic formulations, e.g., formulations of vaccine.

[0043] The present invention further provides NDV chimeric viruses. In particular, the present invention provides a chimeric NDV, comprising a fusion protein, having (i) an ectodomain of a protective antigen from an infectious agent, other than NDV fused to (ii) a cytoplasmic and transmembrane domain of a glycoprotein encoded by an essential gene of an NDV, wherein the fusion protein is incorporated into an NDV, wherein the function of the essential gene is supplied by the fusion protein or glycoprotein native to the NDV. In some embodiments, the essential NDV gene of NDV is the gene encoding an F protein. In other embodiments, the essential NDV gene of NDV is the gene

[0044] The present invention provides a chimeric NDV, comprising a packaged genome comprising a nucleotide sequence encoding an F protein - fusion protein having the cytoplasmic and transmembrane domains of an F protein and the ectodomain of an antigen of an infectious agent, other than NDV, or a disease antigen that is anchored by the C-terminus, so that the F protein - fusion protein is expressed and incorporated into the chimeric NDV. In some embodiments, the chimeric NDV genome comprises a nucleotide sequence encoding an F protein, such that the F protein is expressed and incorporated into the chimeric NDV in addition to the F protein - NDV fusion protein. In other embodiments the nucleotide sequence encoding the F protein - NDV fusion protein replaces the nucleotide sequence encoding the NDV F protein and the F protein - fusion protein supplies the function of the F protein for the chimeric NDV.

[0045] The present invention provides a chimeric NDV, comprising a packaged genome comprising a nucleotide sequence encoding an HN fusion protein having the cytoplasmic and transmembrane domains of an HN protein and the ectodomain of an antigen of an infectious agent, other than NDV, or a disease antigen that is anchored by the N-terminus, so that the HN fusion protein is expressed and incorporated into the chimeric NDV. In some embodiments, the chimeric NDV genome comprises a nucleotide sequence encoding an HN protein, such that the HN protein is expressed and incorporated into the chimeric NDV in addition to the NDV HN fusion protein. In other embodiments, the nucleotide sequence encoding the HN fusion protein replaces the nucleotide sequence encoding the HN protein of NDV and the HN fusion protein supplies the function of the HN protein for the chimeric NDV. The present invention provides recombinant nucleic acid molecules encoding and/or encoding the NDV HN protein or F protein.

[0046] The present invention provides methods for propagating a chimeric NDV of the invention, comprising culturing the chimeric NDV in an embryonated egg or a cell line that is susceptible to NDV infection. The present invention further provides a method for producing an immunogenic formulation, the method comprising: (a) propagating a chimeric NDV of the invention in an embryonated egg or a cell line that is susceptible to NDV infection; and (b) collecting the progeny virus, wherein the virus is grown in sufficient quantities and under sufficient conditions that the virus is free from contamination such that the progeny virus is suitable for use in immunogenic formulations, e.g. vaccine formulations.

[0047] The present invention provides embryonated eggs comprising the chimeric viruses of the invention. The present invention further provides cell lines comprising the chimeric viruses of the invention. The present invention further provides immunogenic formulations comprising the chimeric viruses of the invention.

[0048] The present invention provides methods of inducing an immune response to one, two or more infectious agents in an individual, the method comprising administering an effective amount of a chimeric influenza virus of the invention. In some embodiments, the subject is a human subject. In other embodiments, the subject is a non-human mammal (e.g., a pig, horse, dog, or cat). In still other embodiments, the subject is an avian subject. In a specific embodiment, the present invention provides a method of inducing an immune response to one, two or more infectious agents in an avian, the method comprising administering an effective amount of a chimeric avian influenza virus of the invention.

[0049] The present invention provides methods for inducing an immune response to one, two or more infectious agents in an individual, the method comprising administering to the individual an effective amount of a chimeric NDV of the invention. In some embodiments, the subject is a human subject. In other embodiments, the subject is a non-human mammal (e.g., a pig, horse, dog, or cat). In still other embodiments, the subject is an avian subject. In a specific embodiment, the present invention provides

[0050] The present invention provides methods for inducing an immune response to one, two or more infectious agents in an individual, the method comprising administering to the individual an effective amount of an attenuated chimeric influenza virus of the invention. In some embodiments, the subject is a human subject. In other embodiments, the subject is a non-human mammal (e.g., a pig, horse, dog, or cat). In still other embodiments, the subject is an avian subject. In a specific embodiment, the present invention provides methods for inducing an immune response to one, two or more infectious agents in a human, the method comprising administering to a human in need thereof an effective amount of a chimeric virus of the invention.

[0051] The present invention provides methods for inducing an immune response to a disease antigen, the methods comprising administering to the individual an effective amount of a chimeric virus of the invention. In some embodiments the individual is a human. In other embodiments, the individual is a bird.

3.1 Terminology

[0052] As used herein, the term "animal" includes, but is not limited to, companion animals (e.g., dogs and cats), zoo animals, farm animals (e.g., ruminants, non-ruminants, livestock and chicken), wild animals, and laboratory animals (for example, rodents, such as rats, mice, and guinea pigs, and rabbits), and animals that are cloned or modified either genetically or otherwise (for example, transgenic animals).

[0053] As used herein, the term "about" or "approximately" when used in association with a number refers to any number within 1, 5 or 10% of the referenced number.

[0054] As used herein, the phrase "amino terminus" of NS1 refers to the amino acids from the amino terminal amino acid residue (amino acid residue 1) to amino acid residue 115, amino acid residues 1 to 100, amino acid residues 1 to 75, amino acid residues 1 to 50, amino acid residues 1 to 25, or amino acid residues 1 to 10 of the influenza viral NS1 protein. Amino terminus deletions may include deletions consisting of 5, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165, 170 or 175 amino acid residues from the amino terminus of NS1

[0055] As used herein, the phrase "carboxy terminus" of NS1 refers to amino acid residues 116 to the carboxy terminal amino acid residue, amino acid residues 101 to the carboxy terminal amino acid residue, amino acid residues 76 to the of carboxy terminal amino acid residues, amino acid residues 51 to the carboxy terminal amino acid residue, or amino acid residues 26 to the carboxy terminal amino acid residue of the equine influenza viral NS1 protein, when the amino terminus of NS1 includes amino acid residues 1 to amino acid residue 115, amino acid residues 1 to 100, amino acid residues 1 to 75, amino acid residues 1 to 50, or amino acid residues 1 to 25, respectively, of an influenza viral NS1 protein. Deletions of the carboxy terminus may include deletions consisting of 5, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 73, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165, 170 or 175 amino acid residues from the carboxy terminus of NS1.

[0056] As used herein, the terms "disease" and "disorder" are used interchangeably to refer to a condition in an individual and encompass, but are not limited to, proliferative disorders (e.g., leukemia, fibrosis, carcinoma ( including malignant, non-malignant, metastatic and non-metastatic carcinomas), and lymphoma), and infections by an infectious agent (e.g., a virus, bacteria, parasite), or a condition or symptom associated therewith.

[0057] As used herein, the term "epitopes" refers to sites, fragments or a region of a molecule (e.g., a polypeptide or protein) having antigenic or immunogenic activity in an individual. An epitope having immunogenic activity is a site, fragment or region of a molecule (e.g., polypeptide or protein) that evokes an antibody response in the individual. An epitope having antigenic activity is a site, fragment or region of a molecule to which an antibody binds immunospecifically as determined by any method well known to one skilled in the art, for example by immunoassays.

[0058] As used herein, the term "fragment" in the context of a proteinaceous agent refers to a peptide or polypeptide comprising an amino acid sequence of at least 2 contiguous amino acid residues, at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 residues amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 amino acid residues contiguous, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a peptide, polypeptide or protein. In one embodiment, a fragment of a full-length protein retains activity of a full-length protein. In another embodiment, the full-length protein fragment does not retain the activity of the full-length protein.

[0059] As used herein, the term "fragment" in the context of a nucleic acid encoding a polypeptide or protein refers to a nucleic acid comprising a nucleic acid sequence of at least 2 contiguous nucleotides, at least 5 contiguous nucleotides, at least Less 10 contiguous nucleotides, at least 15 contiguous nucleotides, at least 20 contiguous nucleotides, at least 125 contiguous nucleotides, at least 150 adjoining nucleotides, at least 175 contiguous nucleotides, at least 200 adjoining nucleotides, at least 250 contiguous nucleotides, at least 300 contiguous nucleotides, at least 350 contiguous nucleotides, or at least 380 contiguous nucleotides of the nucleic acid sequence encoding a peptide, polypeptide or protein. In a preferred embodiment, a fragment of a nucleic acid encodes a peptide or polypeptide that retains full-length protein activity. In another embodiment, the full-length protein fragment does not retain the activity of the full-length protein.

[0060] The term "heterologous sequence" as used herein in the context of a proteinaceous agent refers to a molecule that is not found in nature as being associated with the main backbone of chimeric viruses or, in particular, the glycoprotein of viruses chimeric. The term "heterologous sequence" in the context of a nucleic acid sequence or nucleic acid molecule refers to a molecule that is not found in nature as being associated with the chimeric virus backbone genome.

[0061] The term "immunospecifically binds an antigen" and analogous terms as used herein refers to molecules that specifically bind to an antigen and do not specifically bind to another molecule (e.g., antigen-specific antibodies including both modified antibodies ( that is, antibodies that comprise a modified IgG constant domain (e.g., IgG1), or FcRn binding fragment thereof (e.g., Fc domain or Fc-hook domain), and unmodified antibodies (i.e., antibodies that do not comprise a modified IgG constant domain (e.g., IgG1), or FcRn binding fragment thereof (e.g., the Fc domain or Fc hook domain). Molecules that specifically bind an antigen may be cross-reactive with related antigens Preferably, a molecule that specifically binds an antigen does not cross-react with other antigens.A molecule that specifically binds an antigen can be identified, for example, by immunoassays, BIAcore, or other techniques known to those skilled in the art. A molecule that specifically binds an antigen when it binds to said antigen with greater affinity than to any cross-reactive antigen as determined using experimental techniques such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISAs). See, for example, Paul, ed., 1989, Fundamental Immunology 2a. ed., Raven Press, New York on pages 332-336 for a discussion of antibody specificity.

[0062] As used herein, the term "in combination" in the context of administering (a) therapy(s) to an individual, refers to the use of more than one therapy (e.g., more than one prophylactic agent and/or or therapeutic agent). The use of the term "in combination" does not restrict the order in which therapies (e.g., prophylactic and/or therapeutic agents) are administered to the individual (e.g., an individual with an influenza virus infection, and NDV infection, or a condition or symptom associated therewith, or an individual with another infection (e.g., another viral infection)). A first therapy (e.g., a first prophylactic or therapeutic agent) may be administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes , 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) to an individual (e.g., an individual with an influenza virus infection, a NDV or a condition or symptom associated therewith, or another infection (e.g., another viral infection)).

[0063] As used herein, the phrase "interferon antagonistic activity" of a proteinaceous agent refers to a protein or polypeptide, or fragment, derivative, or analogue thereof that reduces or inhibits the cellular interferon immune response. In particular, a protein or polypeptide, or fragment, derivative, or analogue thereof (e.g., NS1 of influenza virus) that reduces interferon antagonistic activity or inhibits interferon expression and/or activity. In a specific embodiment, the phrase "interferon antagonist activity" refers to a virus protein or polypeptide, or fragment, derivative, or analogue thereof (e.g., an influenza virus protein) that reduces or inhibits the response cellular interferon immune system. A viral protein or polypeptide with interferon antagonist activity may preferentially affect the expression and/or activity of one or two types of interferon (IFN). In one embodiment, the expression and/or activity of IFN-a is affected. In another embodiment, the expression and/or activity of IFN-ß is affected. In another specific embodiment, the expression and/or activity of IFN-Y is affected. In some embodiments, the expression and/or activity of IFN-a, IFN-ß and/or IFN-γ in an embryonated egg or cell is reduced from approximately 1 to approximately 100-fold, from approximately 5 to approximately 80-fold, from approximately 20 to approximately 80 times, approximately 1 to approximately 10 times, approximately 1 to approximately 5 times, approximately 40 to approximately 80 times, or 1, 2, 3, 4, 5, 7, 10, 15, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times by a proteinaceous agent with interferon antagonistic activity relative to the expression and/or activity of IFN-a, IFN- ß, and/or IFN-γ in a control embryonated egg or a cell not expressing or not contacted with such a proteinaceous agent as measured by techniques described herein or known to one skilled in the art.

[0064] As used herein, the phrases "IFN-deficient systems" or "IFN-deficient substrates" refer to systems, for example, cells, cell lines and animals, such as mice, chickens, turkeys, rabbits, rats, horses etc., which do not produce one, two or more types of IFN, or which do not produce any type of IFN, or produce low levels of one, two or more types of IFN, or produce low levels of IFN (i.e. a reduced at any IFN expression of 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more when compared to IFN competent systems under the same conditions), do not respond or respond less efficiently to one, two or more types of IFN, or do not respond to any type of IFN, and/or are deficient in the activity of antiviral genes induced by one, two or more types of IFN, or induced by any type of IFN.

[0065] As used herein, the terms "infection", "influenza infection", "avian influenza infection" and "NDV infection" refer to all stages of an influenza virus, an avian influenza virus, an NDV's, or other life cycle infectious agent (e.g., another viral or bacterial infection) in an individual (including, but not limited to, invasion by and replication of influenza virus, avian influenza virus, NDV, or other infectious agent in the cell or tissue of the body), as well as the pathological state resulting from invasion by and replication of influenza viruses, avian influenza viruses, or NDV. Invasion by and multiplication of an influenza virus, avian influenza virus, NDV, or other infectious agent includes, but is not limited to, the following steps: entry of viruses (e.g., influenza virus, avian influenza virus, or NDV) to a cell, fusion of a virus with a cell membrane, the introduction of viral genetic information into a cell, the expression of viral proteins (e.g., proteins from influenza viruses, avian influenza viruses or NDV), the production of new viral particles (i.e., influenza virus, avian influenza virus, or NDV particles) and the release of virus (e.g., influenza virus particles, avian influenza virus, or NDV) from a cell . A respiratory infection (e.g., infection with an influenza virus or NDV) may be an upper respiratory tract infection (URI), a lower respiratory tract infection (LRI), or a combination thereof. In specific embodiments, the infection is a secondary infection (e.g., secondary pneumonia) that manifests after the onset of the primary infection (e.g., viral pneumonia). Secondary infections arise due to a primary infection or a symptom or condition associated with it predisposing the infected individual to such secondary infection. In specific embodiments, the disease state resulting from invasion by and replication of an influenza virus, avian influenza virus, or NDV is an influenza virus, avian influenza virus, or acute NDV disease. Acute stages of respiratory infections may manifest as pneumonia and/or bronchiolitis, where such symptoms may include hypoxia, apnea, shortness of breath, rapid breathing, wheezing, cyanosis, etc. The acute stage of respiratory infections (e.g., infection with influenza viruses and NDVs) requires that an affected individual obtain medical intervention, such as hospitalization, administration of oxygen, intubation, and/or ventilation.

[0066] As used herein, the term "isolate", in the context of viruses, refers to a virus that is derived from a single parental virus. A virus can be isolated using routine methods known to one skilled in the art including, but not limited to, those based on plaque purification and limiting dilution.

[0067] As used herein, the term "isolated" in the context of nucleic acid molecules refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the native source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when synthesized. chemically. In a preferred embodiment, a nucleic acid molecule encoding a viral protein is isolated.

[0068] As used herein, the terms "controlling" "controlling" and "control" refer to the beneficial effects that an individual derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of the condition. disease (e.g. infection). In some embodiments, a subject is administered one or more therapies (e.g., prophylactic and therapeutic agents, such as one of the invention) to "control" an influenza virus infection, avian influenza virus infection, or NDV infection or an infection with other agents

[0069] As used herein, the phrase "multiplicity of infection" or "MOI" is the average number of viruses per infected cell. The MOI is determined by dividing the number of virus added (ml added x Pfu) by the number of cells added (ml added x cells/ml).

[0070] As used herein, the phrase "NS1 gene" refers to the gene encoding the non-structural protein (NS1) in influenza. NS1 is one of eight molecules encoded by the segmented genome of influenza A and other viruses. An "NS1 gene product" refers to a gene product (e.g., an RNA or protein) encoded by an NS1 gene. In the case of a protein, the NS1 gene product is full length and has wild-type NS1 activity (e.g., strain A/WSN/33).

[0071] As used herein, the terms "nucleic acids", "nucleotide sequences" and "nucleic acid molecules" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogues of DNA or RNA molecules. Such analogs can be generated using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogues may also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cell membranes. The nucleic acids or nucleotide sequences may be single-stranded, double-stranded, may contain both single-stranded and double-stranded parts, and may contain triple-stranded parts, but preferably is double-stranded DNA.

[0072] As used herein, the terms "prevent", "preventing" and "prevention" refer to the prevention of a recurrence or onset of, or a reduction in, one or more symptoms of a disease (e.g., viral infection or other infectious diseases) in an individual as a result of administration of a therapy (e.g., a prophylactic or therapeutic agent). For example, in the context of administering therapy to an individual for an infection, "preventing," "preventing," and "preventing" refer to the inhibition of or a reduction in the development or initiation of an infection (e.g., an infection influenza virus, an NDV infection or a condition associated therewith, or an infection other than influenza virus or NDV infection or a condition associated therewith), or the prevention of the recurrence, onset or development of one or more symptoms of an infection (for example, an influenza virus infection, an NDV infection or a condition associated therewith or an infection other than an influenza virus infection, an NDV infection or a condition associated therewith), in an individual resulting from the administration of a therapy (e.g., a prophylactic or therapeutic agent), or from the administration of a combination of therapies (e.g., a combination of prophylactic and therapeutic agents).

[0073] As used herein, the term "protective antigen" in the context of an infectious agent includes any molecule that is capable of eliciting a protective immune response when administered to an individual, whose immune response is directed against the infectious agent.

[0074] As used herein, the terms "prophylactic agent" and "prophylactic agents" refer to any agent(s) that can be used in the prevention of a disease (e.g., an infection) or a symptom thereof (e.g. example, an influenza virus infection, an NDV infection or a condition or symptom associated therewith, or an influenza virus infection other than an NDV infection or a condition or symptom associated therewith). Preferably, a prophylactic agent is an agent that is known to be useful, has been or is being used to prevent or prevent the onset, development, progression and/or severity of a disease or a symptom thereof (e.g., an infection or a condition or a symptom associated with it).

[0075] As used herein, the phrase "purified" in the context of viruses refers to a virus that is substantially free of cellular material and culture media from the cell or tissue source from which the virus is derived. The term "substantially free of cellular material" includes virus preparations in which the virus is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, viruses that are substantially free of cellular material include protein preparations having less than about 30%, 20%, 10%, or 5% (by dry weight) cellular protein (also referred to herein as a "contaminant protein"). ). The virus is also substantially free of culture medium, that is, culture medium represents less than about 20%, 10%, or 5% of the volume of the virus preparation. A virus can be purified using routine methods known to those skilled in the art including, but not limited to, chromatography and centrifugation.

[0076] As used herein, the terms "individual" or "patient" are used interchangeably. As used herein, the terms "individual" and "individuals" refer to an animal (e.g., birds, reptiles, and mammals). In some embodiments, the subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, horse, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey , chimpanzee, and a human). In some embodiments, the subject is a non-human mammal. In other embodiments the individual is a human. In some embodiments, the mammal (e.g., human) is aged 0 to 6 months, 6 to 12 months, 1 to 5 years, 5 to 10 years, 10 to 15 years, 15 to 20 years, 20 to 25 years, 25 to 30 years, 30 to 35 years, 35 to 40 years, 40 to 45 years, 45 to 50 years, 50 to 55 years, 55 to 60 years , from 60 to 65 years old, from 65 to 70 years old, from 70 to 75 years old, from 75 to 80 years old, from 80 to 85 years old, from 85 to 90 years old, from 90 to 95 years old or from 95 to 100 years old. In a specific embodiment, the subject or patient is a bird. In some embodiments, the bird is 0 to 3 months, 3 to 6 months, 6 to 9 months, 9 to 12 months, 12 to 15 months, 15 to 18 months, or 18 to 24 months.

[0077] As used herein, the term "synergistic" in the context of administration or outcome or therapies, refers to a combination of therapies (e.g., prophylactic and therapeutic agents) that is more effective than the additive effects of any two or more simple therapies (e.g., one or more prophylactic or therapeutic agents). A synergistic effect of a combination of therapies (e.g., a combination of prophylactic and therapeutic agents) allows the use of lower dosages of one or more therapies (e.g., one or more prophylactic and therapeutic agents) and/or the administration of less frequency of said therapies to an individual with an illness (e.g., an influenza virus infection, an NDV infection, or a condition or symptom associated therewith, or an infection other than influenza virus infection, NDV infection, or a condition or symptom associated with it). The ability to utilize lower dosages of therapies (e.g., prophylactic and therapeutic agents) and/or administer said therapies less frequently reduces the toxicity associated with administering said therapies to an individual without reducing the effectiveness of said therapies in preventing or treatment of a disease (e.g., an influenza virus infection or a condition or symptom associated therewith, or an infection other than influenza virus infection, NDV infection or a condition or symptom associated therewith). In addition, the synergistic effect may result in improved efficacy of therapies (e.g., prophylactic and therapeutic agents) in preventing, controlling, or treating a disease (e.g., an influenza virus infection, an NDV infection, or a condition or symptoms associated therewith, or an infection other than influenza virus infection, an NDV infection, or a condition or symptom associated therewith). Finally, synergistic effect of a combination of therapies (e.g., prophylactic and therapeutic agents) can avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

[0078] As used herein, the terms "therapies" and "therapy" may refer to any protocol(s), method(s), and/or agent(s) that may be used in prevention , treatment, control, or amelioration of a disease (e.g., cancer, an influenza virus infection, an NDV infection or a condition or symptom associated therewith, or an infection other than influenza virus infection

[0079] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to any agent(s) that can be used in the prevention, treatment, control, or amelioration of a disease (e.g., an infection or a symptom thereof (e.g., an influenza infection, an NDV infection or a condition or symptoms associated therewith, an infection other than influenza virus infection, NDV infection or a condition or symptom associated therewith) Preferably, a therapeutic agent is an agent that is known to be useful for, or has been or is being used for the prevention, treatment, control, or amelioration of a disease or symptom associated therewith (e.g., an infection by influenza, NDV infection or a condition or symptom associated therewith, an infection other than influenza virus infection, NDV infection or a condition or symptom associated therewith).

[0080] As used herein, the terms "treat," "treatment," and "treating" in the context of administering a therapy to an individual for a disease refer to the eradication, reduction, or improvement of symptoms of said disease. With respect to infections (e.g., influenza virus, or NDV virus), treatment refers to the eradication or control of replication of an infectious agent (e.g., a virus), the reduction in the number of an infectious agent (e.g., (e.g., reduction in virus titer), reduction or improvement in the progression, severity, and/or duration of an infection (e.g., an influenza infection, NDV infection, or a condition or symptoms associated therewith, an infection other than influenza virus infection, NDV infection, or a condition or symptom associated therewith), or the amelioration of one or more symptoms resulting from administration of one or more therapies (including, but not limited to administration of one or more prophylactic and therapeutic agents). With respect to cancer, treatment refers to the eradication, removal, modification, or tissue control of primary, regional, or metastatic cancer that results from administration of one or more therapeutic agents of the invention. In some embodiments, such terms refer to minimizing or slowing the spread of cancer resulting from the administration of one or more therapeutic agents of the invention to an individual with such a disease. In other embodiments, such terms refer to the elimination of disease-causing cells.

4. DESCRIPTION OF FIGURES

[0081] FIG. 1. Schematic representation of a hybrid NAf-HN construct

[0082] The construct encodes nucleotides from the 3' non-coding regions of the WSN NA vRNA, the NA coding region corresponds to the cytoplasmic and transmembrane tail domains of the NA protein plus the first amino acid of the NA ectodomain, the coding region of the NDV B1 HN protein (ectodomain only), two sequential stop codons, the untranslated nucleotides of the WSN NA reading frame and the 5' non-coding region of the WSN vRNA.

[0083] FIG. 2. Schematic representation of changes in the polybasic amino acid sequence of HA

[0084] The nucleotide sequence identified as H5N1 HA represents nucleotides 1013-1045 (SEQ ID NO: 13; amino acid sequence SEQ ID NO: 14) of the open reading frame of the influenza A/Vietnam/ HA surface glycoprotein I 203/04 (H5N1). Nucleotides 1026-1038 were replaced by the single nucleotide cytokine using PCR excise and site-directed mutagenesis resulting in the avirulent HA nucleotide sequence (SEQ ID NO: 15; amino acid sequence SEQ ID NO: 16). The sequence change corresponds to the replacement of the 5-amino acid polybasic sequence with the single amino acid threonine.

[0085] FIG. 3. Schematic representation of change in HA nucleic acid sequence

[0086] The sequence identified as avirulent HA represents nucleotides 1013-1033 of the open reading frame of an HA surface glycoprotein based on consensus sequences of avirulent Influenza A/Vietnam/1203/04 (H5N1) HA proteins (SEQ ID NO: 15; amino acid sequence SEQ ID NO: 16). Underlined adenosine residues were replaced such that mutations were synonymous resulting in nucleotide sequence SEQ ID NO: 17 and amino acid sequence SEQ ID NO: 16.

[0087] FIG. 4A-D. Scheme of truncation mutants of pPol1VN1203 NS A. The H5N1 NS gene segment coding region is nucleotides 833. B. The pPol1VN1203 NS1-126 construct has a deletion in the NS gene from nucleotides 379-456 of a region coding, the insertion of 3 stop codons and a BglII restriction site. C. The pPol1VN1203 NS1-99 construct has a deletion in the NS gene from nucleotides 298-456 of a coding region, the insertion of 4 stop codons, a BglII restriction site and a PacI restriction site. D. The pPol1VN1203 NS1-73 construct has a deletion in the NS gene from nucleotides 219-456 of a coding region, the insertion of 4 stop codons, a BglII restriction site and a PacI restriction site.

[0088] FIG. 5. Schematic of pNDV/B1

[0089] The sequences represented are flanked at the 3' end by a T7 promoter and at the 5' end by an HDV ribozyme and T7 terminator. The insertion site between the P and M genes comprises a unique XbaI restriction site.

[0090] FIG. 6. Western Blot Analysis of KGFR Expression in rNDV Chimeric Viruses

[0091] The chimeric viruses rNDV (track 1), rNDV-KGFR (track 2) and rNDV-KGFR/F-CT (track 3) were cultivated in 10-day-old embryonated chicken eggs. Purified viruses subjected to Western blot analysis using a murine anti-KGFR and an anti-mouse HRPO as the primary and secondary antibodies, respectively.

[0092] FIG. 7. Western Blot Analysis of H7HA Expression in Chimeric rNDV Viruses

[0093] The chimeric viruses rNDV (track 1), rNDV-KGFR (track 2) and rNDV-KGFR/FCT (track 3) were cultured in 10-day-old embryonated chicken eggs. Purified viruses subjected to Western Blot analysis using a murine anti-KGFR and an anti-mouse HRPO as the primary and secondary antibodies, respectively.

[0094] FIG. 8. Modification of the rNDV F protein cleavage site

[0095] (A) Schematic representation of the rNDV/B1 genome with two or three amino acid changes in the cleavage site of its F proteins. The peptide bond that is cleaved in the F protein is indicated with a cut. (B) Syncytia formation in CEF cells infected by rNDVs with modified F proteins. CEF cells infected a multiplicity of infections of 0.001, with viruses of rNDV/B1, rNDV/F2aa, and rNDV/F3aa. Viral spread was monitored every 24 hours by immunofluorescence testing.

[0096] FIG. 9. Construction and characterization of the fusogenic rNDV vector expressing HA HPAI H7 protein.

[0097] (A) Schematic representation of rNDV/F3aa chimeric H7 cDNA construction, with the GE/GS, Kozak, and partial H7HA sequences shown (SEQ ID NO:36). (B) Comparison of viral growth kinetics, Log TCID versus Time after inoculation (hours). Square, rNDV/B1; triangle, rNDV/F3aa; bold asteristic, rNDV/B1-H7; asteristic, rNDV/F3aa- H7 chimeric. (C) Expression of WT H7HA protein or chimeric H7HA protein in cells infected with rNDVs. Track 1, placebo infected; track 2, rNDV/F3aa; track 3, rNDV/ Bl- H7; track 4, rNDV/F3aa- H7 chimeric. Row 1 H7a-poultry house; row 2, a-NDV. (D) Incorporation of the H7HA chimeric protein into rNDV virions was increased when compared to that of the H7 WT HA protein. Track 1, rNDV/B1 -H7; rNDV/F3aa-H7 chimeric. Row 1 H7 a-poultry house; row 2, a-NDV.

5. DETAILED DESCRIPTION OF THE INVENTION

[0098] The present invention provides negative-stranded RNA chimeric viruses engineered to express fusion proteins that incorporate into the virion, methods for producing such chimeric viruses, and the use of such viruses, for example as immunogens, in immunogenic formulations, or in tests. vitro. The chimeric viruses of the invention are characterized by displaying, on the surface of the virion, not only virus-associated antigens but also the fusion protein.

[0099] Viruses that can be engineered according to the methods of the invention can be any enveloped virus. In a specific embodiment, viruses that can be engineered in accordance with the methods of the invention have segmented or non-segmented genomes, single-stranded or double-stranded genomes, and express at least one essential glycoprotein (e.g., NA, HA, HN or F) which is incorporated into the virial envelope. Viruses for use in accordance with the methods of the invention may be selected from naturally occurring strains, variants or mutants; mutagenized viruses (e.g., by exposure to UV irradiation, mutagenesis, and/or passage); reassortants (for viruses with segmented genomes); and/or genetically engineered viruses. For example, mutant viruses can be generated by natural variation, exposure to UV irradiation, exposure to chemical mutagenesis, by passage in non-permissive hosts, by reassortment techniques (i.e., by co-infection with an attenuated segment virus with another strain having the desired antigens), and/or by genetic engineering (e.g., using "reverse genetics"). Non-limiting examples of genome-segmented viruses for use in accordance with the methods of the invention include viruses from the family orthomyxoviridae (e.g., influenza virus), buniaviridae (e.g., Bunyamwera), reoviridae, and arenaviridae (e.g., Lassa fever). . Non-limiting examples of viruses with unsegmented genomes for use in accordance with the methods of the invention include coronaviridae (e.g., human coronavirus (SARS)), hepadnaviridae (e.g., hepatitus A, B, or C viruses), herpesviridae (e.g., , herpes simplex virus), poxviridae (e.g., smallpox), rhabdoviridae (e.g., vesicular stomatitis virus (VSV), Sendai virus, and rabies), paramyxoviridae (e.g., measles and respiratory syncytial virus), and filoviridae ( Marburg and Ebola viruses). In some embodiments, the segmented virus is the influenza virus. In other embodiments the unsegmented virus is NDV.

[0100] In some embodiments, the viruses selected for use in the invention are attenuated and/or have defective IFN antagonist activity; that is, they are infectious and can replicate in vivo, but only generate low titers resulting in subclinical levels of infection that are not pathogenic. Viruses can be attenuated by any method known in the art and/or exemplified herein, for example, by engineering the virus to comprise a mutation in the NS1 gene or to comprise a modification in the polybasic amino acid sequence prior to the cleavage site in the HA protein. Such attenuated viruses engineered according to the invention are therefore ideal candidates for immunogenic formulations, for example, live virus vaccines. When administered to an individual, the attenuated chimeric viruses of the invention are capable of generating an immune response and eliciting immunity to both the virus and the non-native or fusion protein. In some embodiments, the non-native protein is derived from a pathogenic element. By extension, administration of such a chimeric virus to an individual generates an immune response and/or immunity to said pathogenic element in addition to the virus.

[0101] The invention also relates to the use of the chimeric virus of the invention in compositions (e.g., immunogenic formulations) for humans or animals (e.g., birds). In particular, chimeric viruses that are attenuated can be used as vaccines against a wide range of viruses and/or diseases. Because the chimeric virus is engineered to express heterologous gene sequences as foreign epitopes on the virion, compositions comprising a chimeric virus of the invention (e.g., vaccine formulations) can be designed for immunization against multiple strain variants, different viruses, or against infectious agents. or completely different disease antigens (e.g., bacteria, parasites, fungi, or tumor-specific antigens) from which the heterologous gene sequences are derived. Many methods can be used to introduce live attenuated virus formulations to a human or animal subject to induce an appropriate immune or cytokine response. These include, but are not limited to, intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes.

5.1 Chimeric influenza virus 5.1.1 Chimeric avian influenza virus comprising a fusion protein incorporated into its virion

[0102] The present invention encompasses the engineering of an avian influenza virus such that a fusion protein is encoded by the genome and, when expressed, is incorporated into the virion. Any avian influenza virus type, subtype or strain that can be engineered to express and incorporate the fusion protein into the avian influenza virion can be selected and used in accordance with the invention including, but not limited to, naturally occurring strains, variants or mutants, mutagenized viruses, reassortants and/or genetically engineered viruses. In a specific embodiment, the avian influenza viruses of the invention are not naturally occurring viruses. In another specific embodiment, the avian influenza viruses of the invention are genetically engineered viruses. Non-limiting examples of avian influenza viruses include influenza A subtypes H5N1, H6N2, H7N3, H9N2, and H10N7.

[0103] Genetic manipulation of the influenza virus requires engineering at least one of the eight viral RNA segments that comprise the viral genome. Genome mutagenesis can be carried out through "reverse engineering" techniques (see section 5.4). The plasticity of the influenza genome is, however, limited in both the number of segments and the length of segments that can be stably integrated into the virus. The overall stability of long insertions is unknown and segments comprising such insertions, or parts thereof, may be lost due to viral assortment after a few generations. Thus, in a preferred embodiment of the invention, the avian influenza virus is engineered so that one of its two main surface proteins is replaced by a fusion protein.

[0104] Therefore, the present invention provides a chimeric avian influenza virus, comprising at least one fusion protein comprising an ectodomain (ED) of an infectious agent protein other than influenza virus and the cytoplasmic (CT) and transmembrane domains ( TM) or the transmembrane domain (TM) of at least one essential influenza virus glycoprotein, wherein at least one fusion protein functionally replaces at least one essential avian influenza virus glycoprotein. In other words, the avian influenza virus serves as the "backbone" that is engineered to express and incorporate into its virion the fusion protein in place of an essential avian influenza virus glycoprotein. Inclusion of the TM and CT domains or TM domain of an influenza virus glycoprotein corresponding to the essential avian influenza virus glycoprotein functionally replaced by the fusion protein allows the fusion protein to incorporate into the avian influenza virus virion. The TM and CT domains or TM domain of the fusion protein can correspond to or be derived from any influenza virus which allows the fusion protein to incorporate into the backbone virion of the avian influenza virus.

[0105] In some embodiments, the TM and CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of a different type, subtype or strain of avian influenza virus from the than the backbone avian influenza virus. In other embodiments, the TM and CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of an influenza virus other than avian influenza viruses. In other embodiments, the TM and CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of the avian influenza virus backbone.

[0106] The avian influenza virion comprises two main surface glycoproteins, hemagglutinin (HA) and neuraminidase (N), both of which comprise a cytoplasmic domain, a transmembrane domain and an ectodomain. Accordingly, in some embodiments, the TM and CT domains of the fusion protein correspond to the TM and CT domains of either the HA protein or an NA protein of an influenza virus. Because the CT domain of HA or NA may not be necessary for incorporation of the fusion protein into the avian influenza virus virion, the fusion protein, in some embodiments, is engineered to contain only the TM domain of HA. or NA. For example, the CT domain of NA has been shown to be unnecessary for proper envelopment of this protein into influenza A viral envelopes (Garcia-Sastre et al, 1995, Virus Res. 37:37-47, incorporated by reference in its entirety ). For this reason, where structural domains corresponding to those of an NA protein are used in creating the fusion protein, the invention encompasses engineering a fusion protein to contain only a TM domain corresponding to an influenza virus NA protein. Accordingly, in one embodiment of the invention, the fusion protein is engineered to contain only one TM domain, which TM domain corresponds to the TM domain of an influenza virus NA protein.

[0107] The TM and CT domains of the HA and NA proteins of influenza virus HA are structurally distinct in that the domains are located at the C-terminus of the HA protein and the N-terminus of the NA protein. In addition to the diversity of orientation for the two domains in each class of surface glycoprotein, the structural domains of HA and CT may comprise as yet unknown differences in functionality dependent on their relative placement within a polypeptide chain. For this reason, when designing the fusion protein to be engineered into the avian influenza virus, the orientation of the ectodomain of the infectious agent to be fused to the TM and CT domains or the TM domain of an influenza virus glycoprotein will guide the selection of the TM and CT domains or the TM domain. For example, where the ectodomain of an infectious agent is anchored by the N-terminus, the TM and CT domains of an influenza virus NA protein can be used.

[0108] HA and NA exhibit competitive activities with respect to cell fusion and release, respectively, which are necessary for virus infectivity and propagation. HA binds to N-acetyl-neuraminic acid (NeuNAc; sialic acid) on a cell surface leading to virus uptake by the host cell, while NA cleaves sialic acid moieties from the cell surface leading to the release of virus from a cell's progeny infected. Disruption of any of these activities results in a non-functional virus. Consequently, to maintain viral competence, where a surface glycoprotein is replaced, its function in the chimeric virus must be supplied by the fusion protein. In one embodiment of the invention, the chimeric avian influenza virus comprises a fusion protein that exhibits neuraminidase activity. In another embodiment of the invention, the chimeric avian influenza virus comprises a fusion protein that exhibits receptor binding activity. In yet another embodiment of the invention, the chimeric avian influenza virus comprises two fusion proteins one of which exhibits neuraminidase activity, and the other exhibits receptor binding activity. In still other embodiments, the chimeric avian influenza virus comprises a fusion protein comprising an epitope of a heterologous infectious agent, any fusion protein exhibiting neuraminidase activity or receptor binding activity. In another embodiment of the invention, the chimeric avian influenza virus comprises a fusion protein that exhibits receptor binding activity. In a specific embodiment, the chimeric avian influenza virus comprises a surface protein containing the ectodomain of the Newcastle disease virus (NDV) HN protein and the TM and CT domains of the Influenza A/WSN/33 NA protein. , ectodomain of HN that exhibits neuraminidase activity. In other embodiments, the chimeric avian influenza virus comprises a surface protein containing the ectodomain of the HA protein of a heterologous influenza virus (e.g., the HA protein H7 or HA protein H9). HA and NA are encoded by separate segments of the viral genome, and replacement of the entire coding region of the native protein eliminates most of the longer boundaries in the sequence encoding the introduced protein.

[0109] In some embodiments, the fusion protein comprises the transmembrane domain plus 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 immediately adjacent residue(s) of the ectodomain of an essential glycoprotein of influenza viruses. For example, in a specific embodiment, the fusion protein comprises the transmembrane domain of an influenza virus NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s). ) immediately adjacent to the ectodomain of the influenza virus NA protein, and the ectodomain, or fragment thereof, of an infectious agent other than influenza virus such that the fusion protein can functionally replace the function of the NA protein. In another specific embodiment, the fusion protein comprises the cytoplasmic and transmembrane domains of an influenza virus NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the influenza virus NA protein that are immediately adjacent to the transmembrane domain of the influenza virus NA protein, and the ectodomain, or fragment thereof, of an infectious agent other than influenza virus such that the fusion protein can functionally replace the NA protein. In another embodiment, the fusion protein comprises the transmembrane domain or cytoplasmic and transmembrane domains of an NA protein, the complete stem domain, or a fragment thereof, of an NA protein preceding its globular head, and the ectodomain, or fragment thereof, of an infectious agent other than influenza virus such that the fusion protein can functionally replace the function of the NA protein. In another specific embodiment, the fusion protein comprises the transmembrane domain of an influenza virus HA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 immediately adjacent residue(s). of the ectodomain of the HA protein of influenza viruses, and the ectodomain, or fragment thereof, of an infectious agent other than influenza viruses such that the fusion protein can functionally replace the function of the HA protein. In another specific embodiment, the fusion protein comprises the cytoplasmic and transmembrane domains of an influenza virus HA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the influenza virus HA protein that are immediately adjacent to the transmembrane domain of the influenza virus HA protein, and the ectodomain, or fragment thereof, of an infectious agent other than influenza virus such that the fusion protein can functionally replace the HA protein.

[0110] In some embodiments, the at least one chimeric avian influenza virus fusion protein of the invention does not comprise the complete ectodomain of a heterologous protein (e.g., it comprises an antigenic fragment of the ectodomain of a protein of an infectious agent heterologous), and may or may not further comprise one or more fragments of the ectodomain of a native essential glycoprotein. Accordingly, in certain embodiments, the ectodomain of the fusion protein may comprise a fragment of the ectodomain of a protein from a heterologous infectious agent. In other embodiments, the ectodomain of the fusion protein may comprise fragments of both a native essential glycoprotein and a protein from a heterologous infectious agent. In embodiments in which the fusion protein replaces an essential surface glycoprotein, the function of the surface glycoprotein must be supplied by the fusion protein, that is, the fusion protein must exhibit the functionality of the surface glycoprotein it is replacing.

[0111] The present invention encompasses nucleotide sequences (i.e., recombinant segments) encoding the fusion proteins described in this Section 5.1.1. In preferred embodiments, the recombinant segments comprising nucleic acids encoding the fusion proteins described in Section 5.1.1 comprise 3' and 5' incorporation signals that are required for major replication, transcription and envelopment of the vRNAs (Fujii et al, 2003 , Proc. Natl. Acad. Sci. USA 100:20022007; Zheng, et al, 1996, Virology 217:242-251, both of which are incorporated herein by reference in their entirety). In a preferred embodiment, the recombinant segments of the invention thus use the 3' and 5' non-coding and/or non-translated sequences of virus segments within the same viral type or strain as the avian influenza virus of structure dorsal. In specific embodiments, the recombinant segments comprise nucleic acids encoding the fusion proteins described in this Section, 5.1.1, which comprise the 3' non-coding region of an influenza virus NA vRNA, the NA coding region corresponding to the CT and TM domains of the NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the NA protein of influenza viruses that are immediately adjacent to the transmembrane domain of the influenza virus NA protein, the untranslated regions of the NA protein reading frame, and the 5' non-coding region of NA vRNA.

[0112] As an alternative to replacing the NA and HA proteins of avian influenza viruses, "reverse genetics" and bicistronic techniques can be used to produce a chimeric influenza virus comprising an ectodomain, or a fragment thereof, of an influenza protein. an infectious agent other than influenza virus and the TM and/or CT domains of an influenza virus. See, for example, U.S. Patent No. 6,887,699. U.S. Patent No. 6,001,634, U.S. Patent No. 5,854,037 and U.S. Patent No. 5,820,871, each of which is hereby incorporated by reference in its entirety. Bicistronic approaches involve inserting the coding region of the fusion protein into the open reading frame of a required virus protein and its stop codon. The insertion is flanked by an IRES and any untranslated signal sequences of the required protein into which it is inserted and must not disrupt the open reading frame, envelope signal, polyadenylation or transcriptional promoters of the required viral protein. Any IRES well known in the art or described herein may be used in accordance with the invention (e.g., the BiP gene IRES, nucleotides 372 to 592 of GenBank database entry HUMGRP78; or the encephalomyocarditis virus (EMCV) IRES ), nucleotides 14302115 from GenBank database entry CQ867238.).Since the function of HA or NA is not being replaced when the bicistronic approach is used, the ectodomain part of the fusion protein is not limited to a protein that provides the function of the substituted HA or NA protein. The ectodomain of such a fusion protein may correspond to any heterologous molecule, or comprise a fragment of any heterologous molecule, including, but not limited to, antigens, disease antigens, and antigens derived from any protein of an infectious agent (e.g., any protective antigen associated with viral, bacterial, or parasitic infectious agents). Non-limiting examples of antigens derived from or associated with infectious agents for use in accordance with the methods of the invention are provided in Section 5.3, infra.

[0113] Replacing a necessary surface protein of the backbone virus or introducing a recombinant segment into the viral genome can attenuate the resulting chimeric virus, that is, the chimeric virus will exhibit weakened replication relative to the wild type. In some embodiments of the invention, attenuation of the chimeric virus is desired so that the chimeric virus remains at least partially infectious and can replicate in vivo, but only generate low titers resulting in subclinical levels of infection that are non-pathogenic. These attenuated chimeric viruses are especially suitable for embodiments of the invention where the virus is administered to an individual to act as an immunogen, for example, a live vaccine. Viruses can be attenuated by any method known in the art and/or exemplified herein, for example, by engineering the virus to comprise a mutation in the NS1 gene or to comprise a modification in the polybasic amino acid sequence prior to the cleavage site in the HA protein ( see U.S. Patent No. 6,468,544; U.S. Patent No. 6,669,943; Li et al, 1999, J. Infect. Dis. 179:1132-1138, each of which is hereby incorporated by reference in its entirety).

[0114] In one embodiment, an attenuated chimeric avian influenza virus of the invention comprises a genome comprising a mutation in the NS1 gene of the avian influenza backbone virus, which is known in other influenza viruses to decrease the ability of the NS1 gene product antagonizes a cellular interferon response. In another embodiment, an attenuated chimeric avian influenza virus of the invention comprises a genome comprising a mutation in the avian influenza backbone virus HA gene, which is known in other influenza viruses to decrease or eliminate the ability of proteases. cells to cleave the protein into its active form and thereby reduce or eliminate induced HA fusion and infectivity. In yet another embodiment, an attenuated chimeric avian influenza virus of the invention comprises a genome comprising a mutation in both the HA gene and the NS1 gene of the avian influenza backbone virus, which are known in other influenza viruses either separately or when combined to reduce or decrease viral activity. Titers of wild-type and attenuated chimeric avian influenza viruses can be determined using any technique well known in the art or described herein, (e.g., hemagglutination tests, plaque tests, egg infectious doses (EID50), infectious doses of tissue culture (TCID50), etc.) and viruses can be propagated under conditions described herein or well known in the art (e.g., in CEF cells, MDCK cells (e.g., in MEM, 10% v/v calf serum (FCS), 1% penicillin/streptomycin at 37°C in a 5% CO2 humidified incubator) or chicken eggs 5.1.2 Attenuated chimeric influenza virus comprising a fusion protein incorporated into its virion

[0115] The present invention encompasses the engineering of an attenuated influenza virus such that a fusion protein is encoded by the genome and, when expressed, is incorporated into the virion. In other words, the invention encompasses the use of an attenuated influenza virus (the parental virus) as the "backbone" that is engineered to express and incorporate the fusion protein into its virion. Any type or strain of attenuated influenza virus including, but not limited to, naturally occurring strains, variants or mutants, mutagenized viruses, reassortants and/or genetically modified viruses may be used as the backbone thereof is engineered to express and incorporate into the its virion the fusion protein. In a specific embodiment, the parental influenza viruses for use according to the invention are not naturally occurring viruses. In another specific embodiment, the parental influenza viruses for use according to the invention are genetically engineered viruses.

[0116] Influenza viruses for use as the backbone viruses according to the invention may naturally have an attenuated phenotype or may be engineered to comprise a mutation associated with an attenuated phenotype, where such a mutation is known in the art or described herein ( for example, a mutation in the viral NS1 protein or viral HA protein). In specific embodiments, the attenuated virus is influenza A. In other embodiments, the attenuated virus is influenza B. In still other embodiments, the attenuated virus is influenza C. Non-limiting examples of influenza viruses that may be engineered in accordance with the invention include Influenza A subtype H10N4, subtype H10N5, subtype H10N7, subtype H10N8, subtype H10N9, subtype Hl INl, subtype Hl 1N13, subtype Hl 1N2, subtype Hl 1N4, subtype Hl 1N6, subtype Hl 1N8, subtype Hl 1N9, subtype H12N1, subtype H12N4, subtype H12N5, subtype H12N8, subtype H13N2, subtype H13N3, subtype H13N6, subtype H13N7, subtype H14N5, subtype H14N6, subtype H15N8, subtype H15N9, subtype H16N3, subtype HlNl, subtype H1N2 , subtype H1N3, subtype H1N6, subtype H1N9, subtype H2N1, subtype H2N2, subtype H2N3, subtype H2N5, subtype H2N7, subtype H2N8, subtype H2N9, subtype H3N1, subtype H3N2, subtype H3N3, subtype H3N4, subtype H3N5, subtype H3N6, subtype H3N8, subtype H3N9 , subtype H4N1, subtype H4N2, subtype H4N3, subtype H4N4, subtype H4N5, subtype H4N6, subtype H4N8, subtype H4N9, subtype H5N1, subtype H5N2, subtype H5N3, subtype H5N4, subtype H5N6, subtype H5N7, subtype H5N8, subtype H5N9, subtype H6N1, subtype H6N2, subtype H6N3, subtype H6N4, subtype H6N5, subtype H6N6, subtype H6N7, subtype H6N8, subtype H6N9, subtype H7N1, subtype H7N2, subtype H7N3, subtype H7N4, subtype H7N5, subtype H7N7, subtype H7N8, subtype H7N9, subtype H8N4, subtype H8N5, subtype H9N1, subtype H9N2, subtype H9N3, subtype H9N5, subtype H9N6, subtype H9N7, subtype H9N8, or subtype H9N9; Influenza B strain Aichi/5/88, strain Akita/27/2001, strain Akita/5/20013 strain Alaska/16/2000, strain Alaska/1777/2005, strain Argentina/69/2001, strain Arizona/146/2005 , strain Arizona/148/2005, strain Bangkok/163/90, strain Bangkok/34/99, strain Bangkok/460/03, strain Bangkok/54/99, strain Barcelona/215/03, strain Beijing/15/84, strain Beijing/184/93, strain Beijing/243/97, strain Beijing/43/75, strain Beijing/5/76, strain Beijing/76/98, strain Belgium/WVl 06/2002, strain Belgium/WV 107/2002 , strain Belgium/WV 109/2002, strain Belgium/WVl 14/2002, strain Belgium/WV 122/2002, strain Bonn/43, strain Brazil/952/2001, strain Bucharest/795/03, strain Buenos Aires/161/ 00), strain Buenos Aires/9/95, strain Buenos Aires/SW16/97, strain Buenos Aires/VL518/99, strain Canada/464/2001, strain Canada/464/2002, strain Chaco/366/00, strain Chaco /R 113/00, strain Cheju/303/03, strain Chiba/447/98, strain Chongqing/3/2000, clinical isolate strain SAl Thailand/2002, clinical isolate strain SA10 Thailand/2002, clinical isolate strain SA101 Philippines/2002 , clinical isolate strain SA101 Philippines/2002, clinical isolate strain SA110 Philippines/2002), clinical isolate strain SA112 Philippines/2002, clinical isolate strain SA113 Philippines/2002, clinical isolate strain SA114 Philippines/2002, clinical isolate strain SA2 Thailand/2002, clinical isolate strain SA20 Thailand/2002, clinical isolate strain S A38 Philippines/2002, clinical isolate strain S A39 Thailand/2002, clinical isolate strain SA99 Philippines/2002, strain CNIC/27/2001, strain Colorado/2597/2004, strain Cordoba /VA418/99, strain Czechoslovakia/16/89, strain Czechoslovakia/69/90, strain Daeku/10/97, strain Daeku/45/97, strain Daeku/47/97, strain Daeku/9/97, strain B/ Du/4/78, strain B/Durban/39/98, strain Durban/43/98, strain Durban/44/98, strain B/Durban/52/98, strain Durban/55/98, strain Durban/56/ 98, strain England/1716/2005, strain England/2054/2005), strain England/23/04, strain Finland/154/2002, strain Finland/159/2002, strain Finland/160/2002, strain Finland/161/ 2002, strain Finland/162/03, strain Finland/162/2002, strain Finland/ 162/91, strain Finland/164/2003, strain Finland/172/91, strain Finland/173/2003, strain Finland/176/2003 , strain Finland/ 184/91, strain Finland/188/2003, strain Finland/190/2003, strain Finland/220/2003, strain Finland/WV5/2002, strain Fujian/36/82, strain Geneva/5079/03, strain Genoa/11/02, strain Genoa/2/02, strain Genoa/21/02, strain Genova/54/02, strain Genova/55/02, strain Guangdong/05/94, strain Guangdong/08/93, strain Guangdong/5/94, Guangdong/55/89 strain, Guangdong/8/93 strain, Guangzhou/7/97 strain, Guangzhou/86/92 strain, Guangzhou/87/92 strain, Gyeonggi/592/2005 strain, Hannover strain /2/90, Harbin strain/07/94, Hawaii strain/ 10/2001, Hawaii strain/1990/2004, Hawaii strain/38/2001, Hawaii strain/9/2001, Hebei strain/19/94, Hebei strain/ 3/94), Henan strain/22/97, Hiroshima strain/23/2001, Hong Kong strain/110/99, Hong Kong strain/1115/2002, Hong Kong strain/112/2001, Hong Kong strain/123/2001 , strain Hong Kong/1351/2002, strain Hong Kong/1434/2002, strain Hong Kong/147/99, strain Hong Kong/156/99, strain Hong Kong/157/99, strain Hong Kong/22/2001, strain Hong Kong/22/89, strain Hong Kong/336/2001, strain Hong Kong/666/2001, strain Hong Kong/9/89, strain Houston/1/91, strain Houston/1/96, strain Houston/2/ 965 strain Hunan/4/72, strain Ibaraki/2/85, strain ncheon/297/2005, strain India/3/89, strain India/77276/2001, strain Israel/95/03, strain Israel/WVl 87/2002 , strain Japan/1224/2005, strain Jiangsu/10/03, strain Johannesburg/1/99, strain Johannesburg/96/01, strain Kadoma/1076/99, strain Kadoma/122/99, strain Kagoshima/15/94, strain Kansas/22992/99, strain Khazkov/224/91, strain Kobe/1/2002, strain, strain Kouchi/193/99, strain Lazio/1/02, strain Lee/40, strain Leningrad/129/91, strain Lissabon/2/90), Los Angeles strain/1/02, Lusaka strain/270/99, Lyon strain/1271/96, Malaysia strain/83077/2001, Maputo strain/1/99, Mar del Plata strain/595/ 99, strain Maryland/1/01, strain Memphis/1/01, strain Memphis/12/97-MA, strain Michigan/22572/99, strain Mie/1/93, strain Milano/1/01, strain Minsk/318 /90, Moscow/3/03 strain, Nagoya/20/99 strain, Nanchang/1/00 strain, Nashville/107/93 strain, Nashville/45/91 strain, Nebraska/2/01 strain, Netherland/801/ strain 90, strain Netherlands/429/98, strain New York/1/2002, strain NIB/48/90, strain Ningxia/45/83, strain Norway/1/84, strain Oman/16299/2001, strain Osaka/1059/ 97, strain Osaka/983/97-V2, strain Oslo/1329/2002, strain Oslo/1846/2002, strain Panama/45/90, strain Paris/329/90, strain Parma/23/02, strain Perth/211 /2001, Peru strain/1364/2004, Philippines strain/5072/2001, Pusan strain/270/99, Quebec strain/173/98, Quebec strain/465/98, Quebec strain/7/01, Roma strain/1/ 03, strain Saga/S 172/99, strain Seoul/13/95, strain Seoul/37/91, strain Shangdong/7/97, strain Shanghai/361/2002), strain Shiga/T30/98, strain Sichuan/379 /99, Singapore strain/222/79, Spain strain/WV27/2002, Stockholm strain/10/90, Switzerland strain/5441/90, Taiwan strain/0409/00, Taiwan strain/0722/02, Taiwan strain/97271/ 2001, Tehran/80/02 strain, Tokyo/6/98 strain, Trieste/28/02 strain, Ulan Ude/4/02 strain, United Kingdom/34304/99 strain, USSR/100/83 strain, Victoria/103 strain /89, Vienna/1/99 strain, Wuhan/356/2000 strain, WV194/2002 strain, Xuanwu/23/82 strain, Yamagata/1311/2003 strain, Yamagata/K500/2001 strain, Alaska/12/96 strain, strain GA/86, strain NAGASAKI/1/87, strain Tokyo/942/96, or strain Rochester/02/2001; Influenza C strain Aichi/1/81, Ann Arbor strain/1/50, Aomori/74 strain, California/78 strain, England/83 strain, Greece/79 strain, Hiroshima/246/2000 strain, Hiroshima/252/2000 strain , Hyogo strain/1/83, Johannesburg/66 strain, Kanagawa strain/1/76, Kyoto/1/79 strain, Mississippi/80 strain, Miyagi/1/97 strain, Miyagi/5/2000 strain, Miyagi/9 strain /96, Nara strain/2/85, New Jersey strain/76, pig strain/Beijing/115/81, Saitama strain/3/2000), Shizuoka strain/79, Yamagata strain/2/98, Yamagata strain/6/ 2000, strain Yamagata/9/96, strain BERLIN/1/85, strain ENGLAND/892/8, strain GREAT LAKES/1167/54, strain JJ/50, strain PIG/BEIJING/10/81, strain PIG/BEIJING/ 439/82), strain TAYLOR/1233/47, or strain CEPA C/YAMAGATA/10/81.

[0117] In one embodiment, the attenuated influenza virus (the parental virus) used according to the invention has a decreased ability to antagonize cellular interferon (IFN). In a specific embodiment, the attenuated influenza virus (the parental virus) used in accordance with the invention is a type or strain of influenza virus comprising a mutation in the NS1 gene that results in an impaired ability of the virus to antagonize influenza. cellular interferon response. Examples of types of mutations that can be introduced into the influenza virus NS1 gene include deletions, substitutions, insertions and combinations thereof. One or more mutations may be introduced anywhere along the NS1 gene (e.g., the N-terminus, the C-terminus or somewhere in between) and/or the regulatory element of the NS1 gene. In a specific embodiment, an attenuated influenza virus (the parental virus) used in accordance with the invention comprises a genome having an influenza virus NS1 gene with an N-terminus mutation. In another embodiment, an attenuated influenza virus (the parental virus) comprises a genome having an influenza virus NS1 gene with a C-terminal mutation. In another embodiment, an attenuated influenza virus (the parental virus) used in accordance with the invention comprises a genome having a mutation in an influenza virus NS1 gene resulting in a deletion consisting of 5, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 99, 100, 105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155, 160, 165, 170, or 175 amino acid residues from the C-terminus of NS1, or a deletion of between 5-170, 25-170, 50-170, 100-170, 100160, or 105 -160 amino acid residues from the C-terminus. In another embodiment, an attenuated influenza virus (the parental virus) used in accordance with the invention comprises a genome having a mutation in an influenza virus NS1 gene resulting in a deletion of all amino acid residues except amino acid residues. 1-126, amino acid residues 1-120, amino acid residues 1-115, amino acid residues 1-110, amino acid residues 1-100, amino acid residues 1-99, amino acid residues 1-95, amino acid residues

[0118] In one embodiment, an attenuated influenza virus of the invention comprises a genome comprising a mutation in the NS1 gene of the influenza virus backbone, which decreases the ability of the NS1 gene product to antagonize an interferon response. cellular, and allows the attenuated virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or an MOI of 0 .0005, 0.0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 , 4.0, 4.5, 5.0, 5.5, or 6.0, grow in titers between approximately 1 to approximately 100 times, approximately 5 to approximately 80 times, approximately 20 to approximately 80 times, or approximately 40 to approximately 80 times, approximately 1 to approximately 10 times, approximately 1 to approximately 5 times, approximately 1 to approximately 4 times, approximately 1 to approximately 3 times, approximately 1 to approximately 2 times, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times lower than wild-type influenza viruses in cells (e.g., cells of human, mouse, rat, guinea pig, dog, horse, or bird origin (e.g., HEp-2, A549, 293T, canine kidney cells) Madin-Darby (MDCK) or chicken embryo fibroblasts (CEF)), as determined at approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3, 4, 5, 6, 7, 8 , 9, 10 days post-infection when propagated under the same conditions. Titers of attenuated and wild-type influenza viruses can be determined using any technique well known in the art or described herein, (e.g., hemagglutination tests, plaque tests, egg infectious doses (EID50), tissue culture infectious doses (TCID50), etc.) and viruses can be propagated under conditions described herein or well known in the art (e.g., in CEF cells, MDCK cells (e.g., in MEM, 10% v/v fetal calf serum(FCS ), 1%

[0119] In another embodiment, the attenuated influenza virus (the parental virus) used according to the invention comprises a genome comprising a mutation in the influenza virus backbone HA gene that decreases or eliminates the ability to protease cells to cleave the protein into its active form. Examples of types of mutations that can be introduced into the influenza A HA gene include deletions, substitutions, insertions or combinations thereof. One or more mutations are preferably introduced into the HA cleavage site (e.g., GenBank nucleotides 1013-1039, entry AY818135). In general, mutations that decrease the cleavability of the HA protein as determined by standard methods in CEF correlate with decreased virulence in in vivo tests (Horimoto and Kawaoka, 1994, 68:3120-3128; which is thus incorporated by reference in its entirety). In a specific embodiment, an attenuated influenza virus (the parental virus) used in accordance with the invention comprises a genome having a mutation in the influenza virus HA gene resulting in the substitution of nucleotides 1026-1038 with the nucleotide thymine single. In another embodiment, an attenuated influenza virus of the invention comprises a genome comprising a mutation in the influenza virus backbone HA gene that decreases or eliminates the ability of cellular proteases to cleave the protein into its active form, and allows the attenuated virus, at a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or an MOI of 0.0005, 0 .0007, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 , 4.5, 5.0, 5.5, or 6.0, grow in titers between approximately 1 to approximately 100 times, approximately 5 to approximately 80 times, approximately 20 to approximately 80 times, or approximately 40 to approximately 80 times , approximately 1 to approximately 10 times, approximately 1 to approximately 5 times, approximately 1 to approximately 4 times, approximately 1 to approximately 3 times, approximately 1 to approximately 2 times, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 times lower than influenza viruses wild-type cells (e.g., cells of human, mouse, rat, guinea pig, dog, horse, or bird origin (e.g., HEp-2, A549, 293T, Madin-Darby canine kidney cells) MDCK) or chicken embryo fibroblasts (CEF)), as determined at approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagated under the same conditions. The HA protein comprising such a mutation is not antigenically distinct from the wild-type parental HA protein, that is, all antibodies raised against the wild-type HA protein will cross-react with the transformed HA protein and all antibodies raised against the transformed HA protein will cross-react with the wild-type HA protein. Titers of attenuated and wild-type influenza viruses can be determined using any technique well known in the art or described herein, (e.g., hemagglutination tests, plaque tests, egg infectious doses (EID50), tissue culture infectious doses (TCID50), etc.) and viruses can be propagated under conditions described herein or well known in the art (e.g., in CEF cells, MDCK cells (e.g., in MEM, 10% v/v fetal calf serum (FCS ), 1% penicillin/streptomycin at 37°C in a 5% CO2 humidified incubator), or embryonated chicken eggs (e.g., in a stationary incubator at 37°C with 55% relative humidity). Alternatively, viruses can be propagated in cells (e.g., CEF cells, MDCK cells, etc.) that are grown in serum-free or serum-reduced (e.g., trypsin TPCK) culture medium.

[0120] In another embodiment, the attenuated influenza virus (the parental virus) used according to the invention comprises a genome comprising: (i) a mutation in the influenza virus backbone HA gene that decreases or eliminates the ability of cellular proteases to cleave the protein into its active form, and (ii) a mutation in the NS1 gene that results in an impaired ability of the virus to antagonize the cellular interferon response. In other embodiments, the attenuated influenza virus of the invention comprises a genome comprising a mutation in both the HA gene and the NS1 gene of the influenza virus backbone that allows the attenuated virus to have a multiplicity of infection (MOI) of between 0.0005 and 0.001, 0.001 and 0.01, 0.01 and 0.1, or 0.1 and 1, or an MOI of 0.0005, 0.0007, 0.001, 0.005, 0.01, 0, 05, 0.1, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0, grow in titers between approximately 1 to approximately 100 times, approximately 5 to approximately 80 times, approximately 20 to approximately 80 times, or approximately 40 to approximately 80 times, approximately 1 to approximately 10 times, approximately 1 to approximately 5 times, approximately 1 to approximately 4 times, approximately 1 to approximately 3 times, approximately 1 to approximately 2 times, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times lower than wild-type influenza viruses in cells (e.g., cells of origin of human, mouse, rat, guinea pig, dog, horse, or poultry (e.g., HEp-2, A549, 293T, Madin-Darby canine kidney (MDCK) or chicken embryo fibroblast (CEF) cells), as determined at approximately 2 to 10 days, 3 to 7 days, 3 to 5 days, or 2, 3, 4, 5, 6, 7, 8, 9, 10 days post-infection when propagated under the same conditions.

[0121] The present invention provides an attenuated chimeric influenza virus, comprising at least one fusion protein having an ectodomain (ED), or fragment thereof, from an infectious agent other than an influenza virus and the cytoplasmic domains (CT) and transmembrane (TM) or the transmembrane domain of an essential influenza virus glycoprotein, wherein the at least one fusion protein functionally replaces at least one essential influenza virus glycoprotein. In other words, the attenuated influenza virus serves as the "backbone" that is engineered to express and incorporate into its virion at least one fusion protein in place of an essential influenza virus glycoprotein. Inclusion of the TM and CT domains or TM domain of an influenza virus glycoprotein corresponding to the essential influenza virus glycoprotein functionally substituted by the fusion protein allows the fusion protein to incorporate into the attenuated influenza virus virion. The TM and CT domains or TM domain of the fusion protein can correspond to or be derived from any influenza virus that allows the fusion protein to incorporate into the backbone virion of the attenuated influenza virus.

[0122] In some embodiments, the TM and CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of a different type, subtype or strain of influenza virus than the attenuated influenza virus with a dorsal structure. In other embodiments, the TM and CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of an influenza virus of a species other than the attenuated backbone influenza virus. . In preferred embodiments, the TM or CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of the attenuated influenza virus backbone.

[0123] In some embodiments, the TM and CT domains of the fusion protein correspond to the TM and CT domains of both an HA protein and an NA protein of an influenza virus. Since the HA or NA CT domain may not be necessary for incorporation of the fusion protein into the influenza virus virion, in some embodiments, the fusion protein is engineered to contain only the HA or NA TM domain. AT.

[0124] The influenza virus TM and CT domains of HA and NA proteins are structurally distinct in that the domains are located at the C-terminus of the HA protein and the N-terminus of the NA protein. In addition to the divergent orientation of the two domains that each class of glycoprotein surfaces, the structural domains of HA and CT may also comprise unknown differences in functionality dependent on their relative placement within a polypeptide chain. Therefore, when designing the fusion protein to be engineered

[0125] To maintain viral competence, where a surface glycoprotein is replaced, its function in the chimeric virus must be supplied by the fusion protein. In one embodiment of the invention, the attenuated chimeric influenza virus comprises a fusion protein that exhibits neuraminidase activity. In another embodiment of the invention, the attenuated chimeric influenza virus comprises the fusion protein that exhibits receptor binding activity. In another embodiment of the invention, the attenuated chimeric virus comprises two fusion proteins, one of which exhibits neuraminidase activity and another of which exhibits receptor binding activity. In yet another embodiment of the invention, the attenuated chimeric influenza virus comprises a fusion protein comprising a fragment of a protein from a heterologous infectious agent, any fusion protein exhibiting neuraminidase activity or receptor binding activity. In a specific embodiment, the attenuated chimeric influenza virus comprises a surface protein containing the ectodomain of the Newcastle Disease Virus (NDV) HN protein and the TM and CT domains of the Influenza A/WSN/33 NA protein. , whose HN ectodomain exhibits neuraminidase activity. In other embodiments, the attenuated chimeric influenza virus comprises a fusion protein containing the HA protein ectodomain of a heterologous influenza subtype or strain (e.g., the H7 HA ectodomain or H9 HA ectodomain).

[0126] In some embodiments, the at least one attenuated chimeric influenza virus fusion protein of the invention does not comprise the complete ectodomain of a heterologous protein (e.g., it comprises an antigenic or protective fragment of the ectodomain of a protein of a heterologous infectious agent), and may or may not further comprise one or more fragments of the ectodomain of a native essential glycoprotein. Accordingly, in certain embodiments, the ectodomain of the fusion protein may comprise a fragment of the ectodomain of a protein from a heterologous infectious agent. In other embodiments, the ectodomain of the fusion protein may comprise fragments of either a native essential glycoprotein or a protein from a heterologous infectious agent. In embodiments in which the fusion protein replaces an essential surface glycoprotein, the function of the surface glycoprotein must be supplied by the fusion protein, that is, the fusion protein must exhibit the functionality of the surface glycoprotein it is replacing.

[0127] The ectodomain of the fusion proteins described in Section 5.1.2 may correspond to or be derived from any glycoprotein, or fragment thereof, of an infectious agent (including viral, bacterial and parasitic infectious agents). Non-limiting examples of infectious agent glycoproteins are provided in Section 5.3, infra.

[0128] In some embodiments, the fusion protein comprises the transmembrane domain plus 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 immediately adjacent residue(s) of the ectodomain of an essential glycoprotein of influenza viruses. In a specific embodiment, the fusion protein comprises the transmembrane domain of an influenza virus NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 immediately adjacent residue(s). of the ectodomain of the NA protein of influenza viruses, and the ectodomain, or fragment thereof, of an infectious agent other than influenza viruses such that the fusion protein can functionally replace the function of the NA protein. In another specific embodiment, the fusion protein comprises the cytoplasmic and transmembrane domains of an influenza virus NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the influenza virus NA protein that are immediately adjacent to the transmembrane domain of the influenza virus NA protein, and the ectodomain, or fragment thereof, of an infectious agent other than influenza virus such that the fusion protein can functionally replace the NA protein. In another embodiment, the fusion protein comprises the transmembrane domain or cytoplasmic and transmembrane domains of an NA protein, the complete stem domain, or a fragment thereof, of an NA protein preceding its globular head, and the ectodomain, or fragment thereof, of an infectious agent other than influenza virus such that the fusion protein can functionally replace the function of the NA protein. In another specific embodiment, the fusion protein comprises the transmembrane domain of an influenza virus HA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 immediately adjacent residue(s). (s) of the ectodomain of the HA protein of influenza viruses, and the ectodomain, or fragment thereof, of an infectious agent other than influenza viruses such that the fusion protein can functionally replace the function of the HA protein. In another specific embodiment, the fusion protein comprises the cytoplasmic and transmembrane domains of an influenza virus HA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the influenza virus HA protein that are immediately adjacent to the transmembrane domain of the influenza virus HA protein, and the ectodomain, or fragment thereof, of an infectious agent other than influenza virus such that the fusion protein can functionally replace the HA protein.

[0129] The present invention encompasses nucleotide sequences (i.e., recombinant segments) encoding the fusion proteins described in this Section 5.1.2. In preferred embodiments, recombinant segments comprising nucleic acids encoding the fusion proteins described in Section 5.1.2 comprise 3' and 5' incorporation signals that are required for proper replication, transcription and envelopment of the vRNAs (Fujii et al. 2003, Proc. Natl. Acad. Sci. USA 100:2002-2007; Zheng, et al, 1996, Virology 217:242-251, both of which are incorporated herein by reference in their entirety). In a preferred embodiment, the recombinant segments of the invention therefore use 3' and 5' non-coding and/or non-translated sequences of virus segments within the same viral type or strain as the structure-attenuated influenza virus. dorsal. In specific embodiments, the recombinant segments comprise nucleic acids encoding the fusion proteins described in Section 5.1.2 that comprise the 3' non-coding region of an influenza virus NA vRNA, the NA coding region corresponding to the domains of CT and TM of the NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the NA protein of influenza viruses that are immediately adjacent to the transmembrane domain of the protein NA of influenza viruses, the untranslated regions of the reading frame of the NA protein and the 5' non-coding region of the NA vRNA. In some embodiments, the recombinant segments comprise nucleic acids encoding the fusion proteins described in Section 5.1.2 that comprise the entire stem domain, or fragment thereof, of an NA protein preceding its globular head.

[0130] As an alternative to replacing the NA and HA proteins of attenuated influenza viruses, "reverse genetics" and bicistronic techniques can be used to produce a chimeric influenza virus comprising an ectodomain of an infectious agent other than influenza virus or an antigen of disease and the TM and/or CT domains of an influenza virus. See, for example, U.S. Patent No. 6,887,699, U.S. Patent No. 6,001,634, U.S. Patent No. 5,854,037 and U.S. Patent No. 5,820,871, each of which is incorporated herein by reference in its entirety. Non-limiting examples of heterologous molecules such as disease antigens and antigens derived from an infectious agent can be used in accordance with the methods of the invention (for example, antigens associated with a disease or viral proteins) are provided in Section 5.3, infra.

5.1.3 Chimeric avian influenza viruses comprising the ectodomain of the HN protein of Newcastle disease virus

[0131] The present invention encompasses the engineering of an avian influenza virus such that a fusion protein comprising the ectodomain of the Newcastle disease virus HN protein is encoded by the genome and, when expressed, is incorporated into the virion. Any type or strain of avian influenza virus that can be engineered to express and incorporate fusion protein into the avian influenza virion can be selected and used in accordance with the invention including, but not limited to, naturally occurring strains, variants or mutants. , mutagenized viruses,

[0132] The present invention provides a chimeric avian influenza virus, comprising a fusion protein having an ectodomain (ED) of a Newcastle disease virus (NDV) HN protein and cytoplasmic (CT) and transmembrane (TM) domains. ) or the transmembrane domain of an influenza virus NA protein, wherein the fusion protein functionally replaces the avian influenza virus NA protein. In other words, the avian influenza virus serves as the "backbone" that is engineered to express and incorporate into its virion the fusion protein in place of the NA protein of avian influenza viruses. Inclusion of the TM and CT domains or TM domain of an influenza virus NA protein in the fusion protein allows the fusion protein to incorporate into the avian influenza virus virion. The TM and CT domains or TM domain of the fusion protein may correspond to or be derived from any influenza virus that allows the fusion protein to be incorporated into the virion of the avian influenza virus backbone.

[0133] The TM and CT domain coding sequences for use according to the invention can be obtained or derived from the published sequence of any NA protein of any influenza subtype or strain (e.g., GenBank, entry AY651447, of strain A /Viet Nam/1203/2004(H5Nl); GenBank, entry AY96877, from strain A/peru/Canada/63 (H6N2); GenBank, entry AY706954, from strain A/duck /Hainan/4/2004 (H6N2); GenBank , entry AY646080, from strain A/chicken/British Columbia/GSC_humano_B/04 (H7N3); or GenBank, entry DQ064434, from strain A/chicken/Beijing/8/98 (H9N2)). In some embodiments, the TM and CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of a different type or strain of avian influenza virus other than avian influenza virus. dorsal structure. In other embodiments, the TM and CT domains or TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of an influenza virus other than avian influenza viruses. In preferred embodiments, the TM and CT domains or the TM domain of the fusion protein correspond to the TM and CT domains or the TM domain of the avian influenza virus backbone. In a specific embodiment, TM and CT domains of the fusion protein correspond to the TM and CT domains of the Influenza A/WSN/33 NA protein.

[0134] In some embodiments, the fusion protein comprises the transmembrane domain of an influenza virus NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) immediately adjacent(s) to the ectodomain of the influenza virus NA protein, and the ectodomain of an NDV HN protein. In another specific embodiment, the fusion protein comprises the cytoplasmic and transmembrane domains of an influenza virus NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the influenza virus NA protein that are immediately adjacent to the transmembrane domain of the influenza virus NA protein, and the ectodomain of an NDV HN protein. In another specific embodiment, the fusion protein comprises the complete stem domain, or a fragment thereof, of an NA protein preceding its globular head and the ectodomain of an NDV HN protein. In other specific embodiments, the fusion protein comprises the transmembrane domain or cytoplasmic and transmembrane domains of an NA protein, and further comprises the entire stem domain, or a fragment thereof, of an NA protein preceding its head. globular and the ectodomain of an NDV HN protein.

[0135] As an alternative to replacing the NA protein of avian influenza viruses, "reverse genetics" and bicistronic techniques can be used to produce a chimeric avian influenza virus comprising an ectodomain of an NDV HN protein and the TM domains and/or CT of an influenza virus. See, for example, U.S. Patent No. 6,887,699, U.S. Patent No. 6,001,634, U.S. Patent No. 5,854,037 and U.S. Patent No. 5,820,871, each of which is incorporated herein by reference in its entirety.

[0136] The present invention encompasses nucleotide sequences (i.e., recombinant segments) encoding the fusion proteins described in this Section 5.1.3. In preferred embodiments, the recombinant segments comprising nucleic acids encoding the fusion proteins described in Section 5.1.3 comprise 3' and 5' incorporation signals that are required for proper replication, transcription and envelopment of the vRNAs (Fujii et al, 2003 , Proc. Natl. Acad. Sci. USA 100:2002-2007; Zheng, et al, 1996, Virology 217:242-251, both of which are incorporated herein by reference in their entirety). In a preferred embodiment, the recombinant segments of the invention therefore use the 3' and 5' non-coding and/or non-translated sequences of virus segments in the same viral type or strain, such as the backbone avian influenza virus. In specific embodiments, the recombinant segment comprises nucleic acids encoding the fusion proteins described in Section 5.1.3, comprises the 3' non-coding region of an influenza virus NA vRNA, an NA coding region corresponding to the CT and TM domains of the NA protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the influenza virus NA protein that are immediately adjacent to the transmembrane domain of the influenza virus NA protein, the untranslated regions of the NA protein reading frame, and the 5' non-coding region of NA vRNA. In another specific embodiment, a recombinant segment comprises, in order from 3' to 5', the 3' non-coding region of WSN NA vRNA (nucleotides 19), nucleotides encoding amino acid residues 1-36 (nucleotides 108). of an NA coding region, nucleotides encoding amino acid residues 51-568 of the NDV B1 HN protein, two sequential stop codons, 157 nucleotides of the WSN NA untranslated reading frame, and the 5' non-coding region of the WSN vRNA (nucleotides 28). See Figure 1.

[0137] Replacement of the backbone influenza virus NA protein or introduction of a recombinant segment into the viral genome can attenuate the resulting chimeric virus, that is, the chimeric virus will exhibit impaired replication relative to the wild type. In some embodiments of the invention, attenuation of the chimeric virus is desired so that the chimeric virus remains at least partially infectious and can replicate in vivo, but only generate low titers, resulting in subclinical levels of infection that are not pathogenic. Such attenuated chimeric viruses are especially suitable for embodiments of the invention where the virus is administered to an individual to act as an immunogen, for example, a live vaccine. Viruses can be attenuated by any method known in the art and/or exemplified herein, for example, by engineering the viruses to comprise a mutation in the NS1 gene or to comprise a modification in the polybasic amino acid sequence prior to the cleavage site in the HA protein ( see U.S. Patent No. 6,468,544; U.S. Patent No. 6,669,943; Li et al, J. Infect. Dis. 179:1132-1138, each of which is incorporated by reference in its entirety).

[0138] In one embodiment, an attenuated chimeric avian influenza virus of the invention comprises a genome comprising a mutation in the NS1 gene of the avian influenza backbone virus, which is known in other influenza viruses to decrease the ability of NS1 gene product to antagonize a cellular interferon response. In another embodiment, an attenuated chimeric avian influenza virus of the invention comprises a genome comprising a mutation in the avian influenza backbone virus HA gene, which is known in other influenza viruses to decrease or eliminate the ability to protease cells to cleave the protein into its active form and thereby reduce or eliminate HA-induced fusion and infectivity. In yet another embodiment, an attenuated chimeric influenza virus of the invention comprises a genome comprising a mutation in both the HA gene and the NS1 gene of the avian influenza backbone virus, which are known in other influenza viruses. influenza either separately or when combined to reduce or decrease viral activity. Titers of wild-type and attenuated chimeric avian influenza viruses can be determined using any technique well known in the art or described herein, (e.g., hemagglutination tests, plaque tests, egg infectious doses (EID50), culture infectious doses (TCID50), etc.) and viruses can be propagated under conditions described herein or well known in the art (e.g., in CEF cells, MDCK cells (e.g., in MEM, 10% v/v fetal calf serum (FCS), 1% penicillin/streptomycin at 37°C in a 5% CO2 humidified incubator), or embryonated chicken eggs (e.g., in a stationary incubator at 370C with 55% relative humidity). can be propagated in cells (e.g., CEF cells, MDCK cells, etc.) that are grown in serum-free or serum-reduced (e.g., trypsin TPCK) culture medium.

5.2 Chimeric Newcastle Disease Virus

[0139] The present invention encompasses the engineering of a Newcastle Disease Virus ("NSV") such that at least one fusion protein is encoded by the genome and, when expressed, is incorporated into the virion. Any type or strain of NDV that can be engineered to express and incorporate at least one fusion protein into the NDV virion can be selected and used in accordance with the invention including, but not limited to, naturally occurring strains, variants or mutants. , mutagenized viruses, reassortants and/or genetically engineered viruses. In a specific embodiment, NDV is a naturally occurring virus. In another specific embodiment, NDV is a genetically engineered virus. For example, as described here, mutant strains of recombinant NDV, rNDV/F2aa and rNDV/F3aa, in which the F protein cleavage site has been replaced with one containing one or two extra arginine residues, allowing the mutant cleavage site to be activated by ubiquitously expressed proteases of the furin family can be used in accordance with the methods of the invention. Non-limiting examples of NDVs that can be used in accordance with the methods of the invention include B1, LaSota, YG97, MET95, and F48E9. In a specific embodiment, the chimeric NDV or rNDV of the invention comprises a fusion protein containing the ectodomain of an influenza A HA protein; In a specific example according to this embodiment, the influenza HA protein is the H7 influenza HA protein.

[0140] The present invention provides a chimeric NDV, comprising at least one fusion protein having an ectodomain (ED), or fragment thereof, of a protein from an infectious agent other than the NDV protein and the cytoplasmic (CT) and /or transmembrane (TM) of an essential NDV glycoprotein. The present invention further provides a chimeric NDV, comprising at least one fusion protein having an ED, or fragment thereof, and TM domain of a protein of an infectious agent other than NDV glycoprotein and the CT of an essential NDV glycoprotein. . The present invention further provides a chimeric NDV, comprising a fusion protein having ED, or fragment thereof, and CT domain of a protein of an infectious agent other than NDV glycoprotein and a TM domain of an essential NDV glycoprotein. In other words, the NDV virus serves as the "backbone" that is engineered to express and incorporate the fusion protein into its virion. Inclusion of the TM and/or CT domains of an essential NDV glycoprotein in the fusion protein allows the fusion protein to incorporate into the NDV virion. The TM and/or CT domains of the fusion protein may correspond to or be derived from any NDV that allows the fusion protein to incorporate into the NDV backbone virion.

[0141] In some embodiments, the TM and/or CT domains of the fusion protein correspond to the TM and/or CT domains of a different type or strain of NDV than the backbone NDV. In preferred embodiments, the TM and/or CT domains of the fusion protein correspond to the TM and/or CT domains of the NDV backbone.

[0142] The NDV virion comprises two main surface glycoproteins: fusion protein (F) and hemagglutinin-neuraminidase (HN), both of which comprise a cytoplasmic domain, a transmembrane domain and an ectodomain. Accordingly, in some embodiments, the TM and/or CT domains of the fusion protein correspond to the TM and/or CT domains of either an F protein or an HN protein of an NDV.

[0143] The TM and CT domains of HN and F protein of NDV and HN protein are structurally distinct in that the domains are located at the C-terminus of the F protein and at the N-terminus of the HN protein. Consequently, when

[0144] In some embodiments, the at least one chimeric NDV fusion protein comprises the TM domain and 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 immediately adjacent residues of the ectodomain of a essential NDV glycoprotein. For example, in a specific embodiment, the fusion protein comprises the transmembrane domain of an NDV F protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) immediately adjacent to the ectodomain of the NDV F protein, and the ectodomain, or fragment thereof, of an infectious agent other than NDV such that the fusion protein can functionally replace the function of the F protein. fusion comprises the cytoplasmic and transmembrane domains of an NDV F protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain of the NDV F protein that are immediately adjacent to the transmembrane domain of the NDV F protein, and the ectodomain, or fragment thereof, of an infectious agent other than NDV such that the fusion protein can functionally replace the F protein. In another specific embodiment, the fusion protein comprises the transmembrane domain of an NDV HN protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 immediately adjacent residue(s) of the ectodomain of the NDV HN protein, and the ectodomain, or fragment thereof, from an infectious agent other than NDV such that the fusion protein can functionally replace the function of the HN protein. In another specific embodiment, the fusion protein comprises the cytoplasmic and transmembrane domains of an NDV HN protein, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 2 or 1 residue(s) of the ectodomain. of the NDV HN protein that are immediately adjacent to the transmembrane domain of the NDV HN protein, and the ectodomain, or fragment thereof, of an infectious agent other than NDV such that the fusion protein can functionally replace the HN protein.

[0145] In some embodiments, a surface NDV glycoprotein (i.e., HN or F protein) is replaced by a fusion protein that supplies the

[0146] In some embodiments, the at least one chimeric NDV fusion protein of the invention does not comprise the complete ectodomain of a heterologous protein (e.g., it comprises an antigenic fragment of the ectodomain of a protein of a heterologous infectious agent), and may or may not further comprise one or more fragments of the ectodomain of a native essential glycoprotein. Accordingly, in some embodiments, the ectodomain of the fusion protein may comprise a fragment of the ectodomain of a protein from a heterologous infectious agent. In other embodiments, the ectodomain of the fusion protein may comprise fragments of both a native essential glycoprotein and a protein from a heterologous infectious agent. In embodiments in which the fusion protein replaces an essential surface glycoprotein, the function of the surface glycoprotein must be supplied by the fusion protein, that is, the fusion protein must exhibit the functionality of the surface glycoprotein it is replacing.

[0147] Since the fusion protein described in this Section 5.2 is not required to replace the function of a necessary viral glycoprotein, the ectodomain of the fusion protein may correspond to or be derived from any heterologous molecule including, but not limited to, any infectious agent antigen (including infectious, viral, bacterial and parasitic agent antigens), and any disease antigen. Non-limiting examples of infectious agent antigens and/or disease antigens are provided in Section 5.3, infra.

[0148] The present invention encompasses nucleotide sequences encoding the fusion proteins described in this Section 5.2. In specific embodiments, a nucleotide sequence comprises nucleic acids encoding a Kozak sequence, followed by the final gene, intercistronic nucleotide (T), and

[0149] In preferred embodiments, the strains of NDV used in accordance with the invention are the lertogenic virus strains, that is, the strains that typically exhibit low virulence or asymptomatic infection in birds, for example, strain B1, strain LaSota or strain Met95. The invention also encompasses the use of highly virulent strains of NDV, for example, YG97 or F48E9, or strains of NDV that have been modified by genetic recombination using methods known in the art or exemplified herein. In a specific embodiment, the invention encompasses the use of an NDV in which the NDV F protein has been genetically modified at the cleavage site in order to increase fusogenic activity. In a specific example according to this invention, the modified F protein comprises two to three amino acid mutations at the F cleavage site. Replacement of a necessary surface protein of the backbone virus or introduction of a nucleotide sequence encoding a protein of fusion in the viral genome may attenuate, or even attenuate, the resulting chimeric virus, that is, the chimeric virus will exhibit impaired replication relative to the wild type. In some embodiments of the invention, attenuation, or re-attenuation, of the chimeric virus is desired as long as the chimeric virus remains at least partially infectious and can replicate in vivo, but only generates low titers resulting in subclinical levels of infection that are non-pathogenic. Such attenuated chimeric viruses are specifically suitable for embodiments of the invention where the virus is administered to an individual to act as an immunogen, for example, a live vaccine. Viruses can be attenuated by any method known in the art.

5.3 Antigens that can be engineered into the chimeric viruses of the invention

[0150] According to the invention, any heterologous molecule can be engineered into the back structure of the virus to elicit an immune response to said molecule. In a specific embodiment, any antigen from any infectious pathogen or associated with any disease that is capable of eliciting an immune response can be engineered into an NDV and/or influenza virus backbone. In a specific embodiment, the antigen is a glycoprotein. In some preferred embodiments, the antigen is capable of functionally replacing an essential glycoprotein of an influenza virus and/or NDV. In specific embodiments, the antigen exhibits neuraminidase or hemagglutinin activities (e.g., receptor binding/fusogenic). In selecting the viral backbone to express the antigen, the orientation of the nucleotide encoding the antigen is considered. For example, where the antigen is naturally anchored via its amino terminus, the TM and CT domains or the TM domain for use in fusion protein engineering will correspond to the TM and CT domains or the TM domain of a viral protein. required from the backbone virus, or related virus, which is also naturally anchored via its amino terminus, for example, the N protein of influenza or the HN protein of NDV.

[0151] In a specific embodiment, a viral antigen is engineered into an NDV or influenza virus backbone. Non-limiting examples of viral antigens include antigens from adenoviridae (e.g., mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, herpes simplex virus 6, Epstein virus -Barr, HHV6-HHV8, and cytomegalovirus), leviviridae (e.g., levivirus, enterobacteria stage MS2, allolevirus), poxviridae (e.g., cordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxvirus, suipoxvirus, moluscipoxvirus, and entomopoxvirinae), papovaviridae ( e.g., polyomavirus and papillomavirus), paramyxoviridae (e.g., paramyxovirus, influenza 1 paravirus, mobilivirus (e.g., measles virus), rubulavirus (e.g., mumps virus), pneumonovirinae (e.g., pneumovirus, respiratory syncytial virus human), human respiratory syncytial virus and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus)), picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus (e.g., human hepatitis A virus), cardiovirus, and apthovirus), reoviridae (e.g., orthoreovirus, orbivirus, rotavirus, cipovirus, fijivirus, phytovirus, and oryzavirus), retroviridae (e.g., mammalian type B retrovirus, mammalian type C retrovirus, avian type C retrovirus, type D, BLV-HTLV retrovirus, lentivirus (e.g., human immunodeficiency virus 1 and human immunodeficiency virus 2 (e.g., HIV gp160), spumavirus), flaviviridae (e.g., hepatitis C virus, dengue virus, West Virus Nile), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphaviruses (e.g., syndbisvirus) and rubiviruses (e.g., rubella virus)), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemeral virus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, Lymphocytic Choriomeningitis Virus, Ippy Virus, and lassa virus), and coronaviridae (e.g., coronavirus and torovirus). In a specific embodiment, the viral antigen is HIV gp120, HIV nef, RSV glycoprotein F, RSV glycoprotein G, influenza neuraminidase virus, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein ( e.g., gB, gC, gD, and gE) or hepatitis B surface antigen, hepatitis C virus E protein, or coronavirus spur protein. In some embodiments, the viral antigen is not gp 41. In some embodiments, the viral antigen is derived from a paramyxovirus. In other, alternative embodiments, the viral antigen is not derived from a paramyxovirus. In some embodiments, the viral antigen is derived from human influenza paravirus type 1, human influenza paravirus type 3, an RSV or Sendai virus. In other, alternative embodiments, the viral antigen is not derived from human influenza paravirus type 1, influenza paravirus type 3, an RSV or Sendai virus. In specific embodiments, the backbone of the virus is an influenza virus and the antigen engineered into the backbone of the influenza virus is not an influenza antigen. In other specific embodiments, the backbone of the virus is an NDV and the antigen engineered into the backbone of the NDV is not an NDV antigen.

[0152] In another embodiment, a bacterial antigen (e.g., bacterial coat protein or protective antigen associated with said bacteria) is engineered into a backbone of NDV or influenza virus. Non-limiting examples of bacterial antigens include antigens from bacteria of the Aquaspirillum family, Azospirillum family, Azotobacteraceae family, Bacteroidaceae family, Bartonella species, Bdellovibrio family, Campylobacter species, Chlamidia species (e.g., Chlamidia pneumoniae), Clostridium, Enterobacteriaceae family (e.g., Citrobacter species, Edwardsiella, Enterobacter aerogenes, Erwinia species, Escherichia coli, Hafnia species, Klebsiella species, Morganella species, Proteus vulgaris, Providencia, Salmonella species, Serratia marcescens, and Shigella flexneri), Gardinella family, Haemophilus influenzae, Halobacteriaceae family, Helicobacter family , Legionallaceae family, Listeria species, Methylococcaceae family, mycobacteria (e.g., Mycobacterium tuberculosis), Neisseriaceae family, Oceanospirillum family, Pasteurellaceae family, Pneumococcus species, Pseudomonas species, Rhizobiaceae family, Spirillum family, Spirosomaceae family, Staphylococcus (e.g., Staphylococcus aureus methicillin-resistant Staphylococcus pyrogenes), Streptococcus (e.g., Streptococcus enteriditis, Streptococcus fasciae, and Streptococcus pneumoniae), Vampirovibr Helicobacter family, Yersinia family, Bacillus anthracis, and Vampirovibrio family.

[0153] Other embodiments, a protective antigen associated with a parasite (e.g., a protozoan is engineered into a dorsal structure of NDV or influenza virus. Any antigen associated with a parasite or protective antigen of a parasite (e.g., a protozoan) can be used in accordance with the methods of the invention. Non-limiting examples of parasite antigens include antigens from a parasite such as an amoeba, a malaria parasite, Plasmodium, Trypanosoma cruzi.

[0154] In another embodiment, a fungal antigen is engineered into a backbone of NDV or influenza virus. Non-limiting examples of fungal antigens include fungal antigens from Absidia species (e.g., Absidia corimbifera and Absidia ramosa), Aspergillus species, (e.g., Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus), Basidiobolus ranarum, Blastomyces dermatitidis, Candida species (e.g., Candida albicans, Candida glabrata, Candida kerr, Candida krusei, Candida parasilosis, Candida pseudo tropicalis, Candida quillermondii, Candida rugosa, Candida stellatoidea, and Candida tropicalis), Coccidioides immitis, species of Conidiobolus, Cryptococcus, Cunninghamella species, dermatophytes, Histoplasma capsulatum, Microsporum gypseum, Mucor pusillus, Paracoccidioides brasiliensis, Pseudallescheria boydii, Rhinosporidium seeberi, Pneumocystis carinii, Rhizopus species (e.g., Rhizopus arrhizus, Rhizopus oryzae, and Rhizopus microsporus), species of Saccharomyces, Sporothrix schenckii, zygomycetes, and classes such as Zygomycetes, Ascomycetes, Basidiomycetes, Deuteromycetes, and Oomycetes.

[0155] In another embodiment, a tumor-associated antigen is engineered into an NDV or influenza virus backbone. Any associated tumor antigen known in the art can be used in accordance with the methods of the invention. Non-limiting examples of tumor-associated antigens include MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, N-acetylglucosaminyltransferase-V, p-15, MART-1/MelanA, TRP-1 (gp75), Tyrosinase, cyclin-dependent kinase 4, ß-catenin, MUM-I, CDK4, HER-2/neu, human papillovirus- E6, human papillovirus E7, MUC-1, caspase-8, CD5, CD20, CEA, mucin- 1 , Lewisx, CA-125, epidermal growth factor receptor, pl85HER2, IL-2R, Fap-a, tenascin, antigens associated with a metalloproteinase, and CAMPATH-1.

5.4 Construction and propagation of chimeric viruses of the invention

[0156] The chimeric viruses of the invention can be generated using the reverse genetics technique. The reverse generic technique involves the preparation of synthetic recombinant viral RNAs that contain the non-coding regions of the negative strand, viral RNA that are essential for recognition by viral polymerases and for envelope signals necessary to generate a mature virion. Recombinant RNAs are synthesized from a recombinant DNA template and reconstituted in vitro with purified viral polymerase complex to form recombinant ribonucleoproteins (RNPs) that can be used to transfect cells. More efficient transfection is performed if viral polymerase proteins are present during transcription of the synthetic RNAs either in vitro or in vivo. Synthetic recombinant RNPs can be rescued into infectious virus particles. The above techniques are described in U.S. Patent No. 5,166,057 issued November 24, 1992; in U.S. Patent No. 5,854,037 issued December 29, 1998; in European Patent Publication EP 0702085A1. published on February 20, 1996; in U.S. Patent Application Series No. 09/152,845; in PCT International Patent Publications WO97/12032 published April 3, 1997; WO96/34625 published November 7, 1996; in European Patent Publication EP A780475; WO 99/02657 published January 21, 1999; WO 98/53078 published November 26, 1998; WO 98/02530 published January 22, 1998; WO 99/15672 published April 1, 1999; WO 98/13501 published April 2, 1998; WO 97/06270 published February 20, 1997; and EPO 780 475A1 published June 25, 1997, each of which is incorporated by reference herein in its entirety.

[0157] Auxiliary-free plasmid technology can also be used to engineer a chimeric virus of the invention. Briefly, with respect to influenza viruses, full-length cDNAs of viral segments are amplified using PCR with primers that include unique restriction sites, which allow insertion of the PCR product into a plasmid vector (Flandorfer et al, 2003, J Virol. 77:9116-9123; Nakaya et al, 2001, J. Virol. 75:11868-11873; both being incorporated herein by reference in their entirety). The plasmid vector is designed to position the PCR product between a truncated human RNA polymerase I promoter and a hepatitis delta virus ribozyme sequence such that an exact negative transcript (sense vRNA) is produced from the polymerase promoter. I. Separate plasmid vectors comprising each viral segment as well as expression vectors comprising required viral proteins are transfected into cells leading to the production of recombinant viral particles. For a detailed description of helper-free plasmid technology see, for example, International Publication No. WO 01/04333; U.S. Patent No. 6,649,372; Fodor et al., 1999, J. Virol. 73:9679-9682; Hoffmann et al, 2000, Proc. Natl. academic Sci. USA 97:6108-6113; and Neumann et al, 1999, Proc. Natl. academic Sci. USA 96:9345-9350, which are incorporated herein by reference in their entirety. Similarly, with respect to the NDV single-segment genome, a complete cDNA from the Hitchner B1 strain was constructed, inserted into a plasmid vector, and engineered to contain a unique restriction site between the P and M genes. according to the invention can then be inserted into the viral genome at the unique restriction site. The unique segment was positioned between a T7 promoter and the hepatitis delta virus ribozyme to produce an exact negative T7 polymerase transcript. The plasmid vector and expression vectors comprising the necessary viral proteins are transfected into cells leading to the production of recombinant viral particles (see Swayne et al, 2003, Avian Dis. 47:1047-1050 and Swayne et al, 2001, J. Virol 11868-11873, each being incorporated by reference in its entirety).

[0158] The chimeric influenza viruses of the invention can be engineered to contain RNA segments that are bicistronic. Bicistronic techniques allow the engineering of multiple protein coding sequences into a single mRNA through the use of IRES sequences. IRES sequences direct the internal recruitment of ribozomes to the RNA molecule and allow downstream translation in a termination-independent manner. Briefly, a coding region of one protein is inserted into the ORF of a second protein. The insertion is flanked by IRES and any untranslated signal sequences necessary for proper expression and/or function. The insertion must not disrupt the open reading frame, polyadenylation or transcriptional promoters of the second protein (see e.g., Garcfa-Sastre et al, 1994, J. Virol. 68:6254-6261 and Garcia-Sastre et al, 1994 Dev Biol Stand 82:237-246), each of which is incorporated herein by reference in its entirety.

5.4.1 Propagation of chimeric viruses

[0159] The chimeric influenza viruses of the present invention can be propagated on any substrate that allows the virus to grow in titles that allow the uses of the chimeric viruses described here. In one embodiment, the substrate allows the chimeric viruses to grow at titers comparable to those determined by the corresponding wild-type viruses. In a specific embodiment, the attenuated chimeric influenza viruses of the invention are propagated on IFN-deficient substrates.

[0160] The chimeric viruses of the invention can be cultivated in cells (e.g., bird cells, chicken cells, etc.) that are susceptible to infection by the viruses, embryonated eggs or animals (e.g., birds). Such methods are well known to those skilled in the art. In a specific embodiment, cells used to propagate attenuated influenza viruses with reduced interferon antagonist activity are deficient in IFN. In one embodiment, the avian chimeric viruses of the invention are propagated in chicken cells or embryonated eggs. Representative chicken cells include, but are not limited to, chicken embryo fibroblasts or chicken embryo kidney cells.

[0161] Chimeric viruses of the invention can be propagated in embryonated eggs, for example, 6 to 14 days old. Young or immature embryonated eggs can be used to propagate the attenuated chimeric influenza viruses of the invention. Immature embryonated eggs encompass eggs that are younger than 10 days old, for example, 6 to 9 day old eggs that are INF deficient. Immature embryonated eggs may also encompass eggs that artificially imitate even immature eggs, which are younger than ten days old, as a result of changes to growth conditions, for example, changes in incubation temperatures; drug treatment; or any other change that results in an egg with delayed development, such that the IFN system is not fully developed compared to a ten to twelve day old egg. The chimeric viruses of the invention can be propagated in different locations of the embryonated egg, for example, the allantoic cavity. For a discussion of growing and propagating viruses, in particular attenuated influenza viruses with reduced interferon antagonist activity see, for example, U.S. Patent No. 6,852,522 and U.S. Patent No. 6,852,522, both of which are incorporated herein by reference in its entirety.

[0162] For virus isolation, the chimeric virus is removed from cell culture and separated into cellular components, typically by

[0163] The chimeric viruses of the invention can be used in active immunization in an individual. In one aspect, the chimeric viruses of the invention can be used to prevent, control and/or treat one or more diseases. In a specific aspect, the chimeric viruses of the invention can be used to prevent, control and/or treat infections by two infectious agents. See Section 5.5.1 for a description of immunogenic formulation and uses of those formulations to induce an immune response in an individual. The chimeric viruses of the invention can also be used to produce antibodies that can be used in diagnostic immunotests, passive immunotherapy, and generation of anti-idiotypic antibodies. For example, a chimeric influenza virus comprising a fusion protein having an ectodomain from an infectious agent other than influenza viruses can be administered to an individual (e.g., a mouse, rat, horse, donkey, bird, or human) to generate antibodies to both the influenza backbone and the infectious agent that can then be isolated and used in diagnostic tests, passive immunotherapy, and generation of antiidiotypic antibodies. The antibodies generated can be isolated by standard techniques known in the art (e.g., immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunotests, passive immunotherapy and generation of anti-idiotypic antibodies. Antibodies isolated before being used in passive immunotherapy can be modified, for example, antibodies can be chimerized or humanized. See, for example, U.S. Patent Nos. 4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741, each of which is incorporated herein by reference in its entirety, for studies on the generation of chimeric and humanized antibodies.

[0164] For antibodies produced by chimeric viruses for use in passive immunization, the dosage administered to an individual is typically 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg at 0 .25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0, 01 to 0.10 mg/kg of the individual's body weight. The antibodies encompassed by the invention may be administered with other prophylactic or therapeutic compositions for de novo immunization or treatment, control or prevention of an infectious condition or disease, or symptom thereof. Administration of antibody doses of the invention may be by bolus injection or delivered more slowly by IV (e.g., over about 5 minutes, about 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 4 hours, or about 6 hours). Dosages of the antibodies of the invention can also be repeated (for example, every day, every 2 days, every 3 days, every week, every 2 weeks, every 3 weeks, every 6 weeks, every 9 weeks, every 12 weeks, every 4 months, every 6 months, every 12 months, every 18 months, or every 2 years) during the course of treatment (e.g., 2 weeks, 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 16 months, 20 months, or 24 months or more). In some embodiments, antibodies produced by the chimeric virus of the invention may be administered parenterally, for example, intravenously, intramuscularly or subcutaneously, or, alternatively, they are administered orally or intranasally. The antibodies encompassed by the invention can also be administered as a sustained release formulation.

[0165] Antibodies isolated from individuals administered a chimeric virus of the invention can also be used to monitor treatment and/or disease progression. Any immunotest system known in the art can be used for this purpose including, but not limited to, competitive and non-competitive testing systems using techniques such as radioimmunoassays, ELISA (

[0166] Antibodies generated by the chimeric viruses of the invention can also be used in the production of anti-idiotypic antibodies. The anti-idiotypic antibody can then, in turn, be used for immunization to produce a subpopulation of antibodies that bind an initial chimeric influenza antigen (Jerne, 1974, Ann. Immunol. (Paris) 125c:373; Jerne et al , 1982, EMBO J. 1:234).

[0167] In immunization procedures, the amount of immunogen to be used and the immunization schedule will be determined by a clinician skilled in the art and will be administered by reference to the individual's immune response and antibody titers.

5.5.1 Immunogenic formulations

[0168] The invention also encompasses the use of the chimeric viruses of the invention in immunogenic formulations, for example, vaccine formulations. In cases where the immunogenic formulations comprise a chimeric influenza virus, the formulations can be used in methods of preventing, controlling, neutralizing, treating and/or ameliorating influenza virus infection, and/or infections by another infectious agent and/or a disease. In cases where the immunogenic formulations comprise a chimeric NDV, the formulations can be used in methods of preventing, controlling, neutralizing, treating and/or ameliorating an NDV infection, infections by another infectious agent and/or a disease.

[0169] Immunogenic formulations may comprise any live or deactivated chimeric virus of the invention. The chimeric virus can be deactivated by methods known to those skilled in the art. Common methods use formalin and heat for inactivation. See, for example, U.S. Patent No. 6,635,246, which is incorporated herein by reference in its entirety. Other methods include those described in U.S. Patent Nos. 5,891,705; 5,106,619 and 4,693,981, incorporated herein by reference in their entirety.

[0170] A live immunogenic formulation may be preferred because multiplication in the individual leads to a prolonged stimulus of a similar type and magnitude to that occurring in natural infections and thus confers lasting and substantial immunity. Production of such live recombinant immunogenic formulations can be achieved using conventional methods involving propagation of the chimeric virus in cell culture or in embryonated eggs (e.g., embryonated chick eggs) followed by purification. Furthermore, chimeric viruses can induce a robust IFN response that has other biological consequences in vivo, conferring protection against subsequent infections.

[0171] In a preferred embodiment, the immunogenic formulations of the present invention comprise an effective amount of a chimeric virus of the invention, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a Federal or State Government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. The term "vehicle" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition (e.g., immunogenic or vaccine formulation) is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable formulations. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, starch, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. The formulation must serve the mode of administration. The particular formulation may also depend on whether the chimeric virus is live or inactivated.

[0172] The immunogenic formulations of the invention can be administered to a native individual, that is, an individual who does not have a disease or has not been or is not currently infected with one or both infectious agents. In one embodiment, the immunogenic formulations are administered to a native individual, that is, an individual who does not have a disease or has not been or is not currently infected with one or both infectious agents, but is at risk of acquiring this disease ( for example, a viral infection). In one embodiment, the immunogenic formulations of the invention are administered to an individual who does not have a disease, or has not been and is not infected with one of the infectious agents so that the chimeric virus induces an immune response. In another embodiment, the immunogenic formulations of the invention are administered to an individual who does not have and is infected with both infectious agents so that the chimeric virus induces an immune response. The immunogenic formulations of the invention can also be administered to an individual who is and/or has been infected with one or both of the infectious agents or another type, subtype or strain of agents to which the chimeric virus induces an immune response.

[0173] Many methods can be used to introduce the immunogenic formulations, for example, vaccine formulations described above, those include but are not limited to intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival and subcutaneous routes. In birds, methods may also include choanal inoculation. As an alternative to parenteral administration, the invention also encompasses mass administration routes for agricultural purposes such as through drinking water or a spray. It may be preferred to introduce the chimeric influenza virus immunogenic formulation through the natural route of wild-type virus infection. Alternatively, it may be preferable to introduce the chimeric virus of the invention through the natural route of infection of the agent from which the fusion protein is derived. The ability of chimeric viruses to induce a vigorous cellular and secretory immune response can be used advantageously. For example, respiratory tract infection by chimeric viruses can induce a strong secretory immune response, for example, in the urogenital system, with concomitant protection against a particular disease-causing agent. In addition, in a preferred embodiment it may be desirable to introduce the pharmaceutical formulations of the invention into the lungs by any appropriate route. Pulmonary administration can also be used, for

[0174] In some embodiments, an immunogenic formulation of the invention does not result in complete protection from an infection (e.g., a viral infection or infection by a non-viral infectious agent), but results in a lower titer or reduced number of the pathogen (e.g. a virus) compared to an untreated individual. In some embodiments, administration of immunogenic formulations of the invention results in a reduction of 0.5-fold, 1-fold, 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50 times, 75 times, 100 times, 125 times, 150 times, 175 times, 200 times, 300 times, 400 times, 500 times, 750 times, or 1,000 times or greater in the pathogen titer relative to an untreated individual. Benefits of a reduction in the titer, number or total pathogen load include, but are not limited to, less severity of symptoms of the infection and a reduction in the extent of the disease or condition associated with the infection.

[0175] In some embodiments, an immunogenic formulation of the invention is used to protect against a disease (e.g., an infection) in native individuals. In a specific embodiment, an immunogenic formulation of the invention is used to protect against an infection with an influenza virus and/or at least one other infectious agent that is not an influenza virus and/or protect against a disease or symptom associated with infection in a native individual. In other embodiments, an immunogenic formulation of the invention is used to protect against infection by NDV and/or at least one other infectious agent and/or protect against a disease or symptom associated therewith in native individuals. Non-limiting examples of such other infectious agents are papillomavirus, herpesvirus, retrovirus (e.g., HIV), hepatitis virus, rhinovirus, respiratory syncytial virus, NDV, cytomegalovirus, adenovirus, Clostridia sp., Salmonella sp., Staphylococci sp., Enterococci sp., Vibrio sp., E.coli, Streptococcus equi, Mycoplasma pneumoniae, Klebsiella pneumoniae and Pseudomonas aeruginosa, and Dermatophilus congolensis, or a protozoan such as amoeba, malaria parasite or Trypanosoma cruzi.

[0176] The prophylactic and/or therapeutic effect of the immunogenic formulations of the invention are based, in part, on obtaining or inducing an immune response (for example, a humoral immune response). In one aspect, the immunogenic formulations induce a detectable serum titer of an antibody against chimeric virus antigens in or in an individual or animal model (e.g., mouse, rat, or canine model). The serum titer of an antibody can be determined using techniques known to one skilled in the art, for example, immunoassays such as ELISAs. In one embodiment, the antibodies specifically bind to an antigen on the backbone of the chimeric virus. In other embodiments, the antibodies specifically bind to an antigen of at least one fusion protein, that is, an antigen of the ectodomain of the introduced protein associated with an infectious agent or disease. In a specific embodiment, the antibodies generated by administering an immunogenic formulation of the invention are neutralizing antibodies.

[0177] In one embodiment, administration of a chimeric virus of the invention to an individual or animal model thereof results in a serum titer of about 1 µg/ml, about 2 µg/ml, about 5 µg/ml ml, about 6 µg/ml, about 10 µg/ml, about 15 µg/ml, about 20 µg/ml, about 25 µg/ml, about 50 mg/ml, about 75 mg/ml , about 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275 mg/ml, or about 300 mg/ml or more of an antibody that specifically binds to an antigen on the backbone of the chimeric virus. In other embodiments, administration of a chimeric virus of the invention to a subject or animal model thereof results in a serum titer of about 1 µg/ml, about 2 µg/ml, about 5 µg/ml, about 6 µg/ml, about 10 µg/ml, about 15 µg/ml, about 20 µg/ml, about 25 µg/ml, about 50 mg/ml, about 75 mg/ml, about 100 mg /ml, about 125 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275 mg/ml ml, or about 300 mg/ml or more of an antibody that specifically binds to a fusion protein antigen, that is, an ectodomain antigen of the introduced protein associated with an infectious agent or disease. Preferably a serum titer of about 1 µg/ml, about 2 µg/ml, about 5 µg/ml, about 6 µg/ml, about 10 µg/ml, about 15 µg/ml, about 20 µg/ml, about 25 µg/ml, about 50 mg/ml, about 100 mg/ml, about 150 mg/ml or about 300 mg/ml or more of such antibodies is achieved in approximately 20 days ( preferably 25, 30, 35 or 40 days) after administration of the first dose of an immunogenic formulation of the invention and without administration of any other doses of the formulation. The immune response can be determined in the individual or in an animal model, such response is correlated or extrapolated to a predicted response in an individual, for example, a human.

[0178] In one embodiment, the present invention provides methods for preventing at least one disease (e.g., an influenza infection and/or infections by another infectious agent other than influenza) in an individual, the methods comprising administering to the said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric influenza virus of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml , 100 µg/ml or greater of antibodies that immunospecifically bind to an antigen or epitope on the backbone of the chimeric virus 2 days, 5 days, 10 days, 15 days, 20 days, or preferably 30 days after the first administration and before to any other subsequent administration. In another embodiment, the present invention provides methods for preventing at least one disease (e.g., an influenza infection and/or infections by another infectious agent other than influenza) in an individual, the methods comprising administering to said individuals a first dose of an effective amount of an immunogenic formulation comprising a chimeric influenza virus of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg /ml or greater of antibodies that immunospecifically bind to a fusion protein antigen (i.e., an ectodomain antigen of the introduced protein associated with the disease) in 2 days, 5 days, 10 days, 15 days, 20 days or, preferably 30 days after the first administration and prior to any subsequent administration. The immune response can be determined in the individual or in an animal model, any response then being correlated or extrapolated to a predicted response in the individual, for example, a human. In one embodiment, the present invention provides methods for preventing an avian influenza infection and/or infections by an infectious agent other than avian influenza in a bird, the method comprising administering a first dose of an immunogenic formulation comprising a chimeric virus. of the avian influenza virus of the invention, which chimeric avian influenza virus comprises a fusion protein containing a heterologous protein sequence, to said individual of an effective amount of the avian chimeric virus of the invention, wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or of antibodies that bind immunospecifically to a chimeric virus antigen and/or antibodies that bind immunospecifically to a fusion protein antigen 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after first administration and prior to any subsequent administration. In some embodiments, the dose of chimeric influenza virus administered to the subject or animal model is 102.5 x 102, 103.5 x 103, 104.5 x 104, 105.5 x 105, 106.5 x 106, 107, 5 x 107, 10s, 5 x 10s, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu.

[0179] In one embodiment, the present invention provides methods for treating at least one disease (e.g., an influenza infection and/or infections by another infectious agent other than influenza) in an individual, the methods comprising administering to the said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric influenza virus of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml , 100 µg/ml or greater of antibodies that immunospecifically bind to an antigen or epitope on the backbone of the chimeric virus 2 days, 5 days, 10 days, 15 days, 20 days, or preferably 30 days after the first administration and before to any subsequent administration. In another embodiment, the present invention provides methods for treating at least one disease (e.g., an influenza infection and/or infections with another infectious agent other than influenza) in an individual, the methods comprising administering to said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric influenza virus of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg /ml or greater of antibodies that immunospecifically bind to a fusion protein antigen (i.e., an ectodomain antigen of the introduced protein associated with the disease) at 2 days, 5 days, 10 days, 15 days, 20 days or, preferably 30 days after the first administration and prior to any subsequent administration. The immune response can be determined in an individual or in an animal model, any response then being correlated or extrapolated to a predicted response in the individual, for example, a human. In one embodiment, the present invention provides methods for treating an avian influenza infection and/or infections by another infectious agent other than avian influenza in a bird, the method comprising administering a first dose of an immunogenic formulation comprising a chimeric virus. of the avian influenza virus of the invention, which chimeric avian influenza virus comprises a fusion protein containing a heterologous protein sequence, to said individual of an effective amount of the avian chimeric virus of the invention, wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or of antibodies that bind immunospecifically to a chimeric virus antigen and/or antibodies that bind immunospecifically to a fusion protein antigen 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after first administration and prior to any subsequent administration. In some embodiments, the dose of chimeric influenza virus administered to the subject or animal model is 102.5 x 102, 103.5 x 103, 104.5 x 104, 105.5 x 105, 106.5 x 106, 107.5 x 107, 108.5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011.5 x 1011 or 1012 pfu.

[0180] In one embodiment, the present invention provides methods for controlling and/or ameliorating at least one disease (e.g., an influenza infection and/or infections by another infectious agent other than influenza) in an individual, the methods comprising administering to said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric influenza virus of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or greater of antibodies that immunospecifically bind to an antigen or epitope on the backbone of the chimeric virus 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after first administration and prior to any subsequent administration. In another embodiment, the present invention provides methods for controlling and/or ameliorating at least one disease (e.g., an influenza infection and/or infections with another infectious agent other than influenza) in an individual, the methods comprising administering to said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric influenza virus of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml , 100 µg/ml or greater of antibodies that bind immunospecifically to a fusion protein antigen (i.e., an antigen from the ectodomain of the introduced protein associated with the disease) at 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after the first administration and prior to any subsequent administration. The immune response can be determined in an individual or in an animal model, any response then being correlated or extrapolated to a predicted response in the individual, for example, a human. In one embodiment, the present invention provides methods for controlling and/or ameliorating an avian influenza infection and/or infections by an infectious agent other than avian influenza in a bird, the method comprising administering a first dose of an immunogenic formulation. comprising a chimeric avian influenza virus of the invention, which chimeric avian influenza virus comprises a fusion protein containing a heterologous protein sequence, to said individual of an effective amount of the avian chimeric virus of the invention, wherein the effective amount is the amount which results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or greater of antibodies that bind immunospecifically to a chimeric virus antigen and/or antibodies that bind immunospecifically to a fusion protein antigen 2 days, 5 days, 10 days, 15 days, 20 days, or, preferably, 30 days after the first administration and before any subsequent administration. In some embodiments, the dose of chimeric influenza virus administered to the subject or animal model is 102.5 x 102, 103.5 x 103, 104.5 x 104, 105.5 x 105, 106.5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu.

[0181] In one embodiment, the present invention provides methods for preventing at least one disease (e.g., an NDV infection and/or infections by another infectious agent that is not NDV) in an individual, the methods comprising administering to the said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric NDV of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount which results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or greater of antibodies that immunospecifically bind to an antigen or epitope on the backbone of the chimeric virus 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after the first administration and prior to any subsequent administration. In another specific embodiment, the present invention provides methods for preventing at least one disease (e.g., an NDV infection and/or infections by another infectious agent other than NDV) in an individual, the methods comprising administering to said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric NDV of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml ml or greater of antibodies that immunospecifically bind to a fusion protein antigen (i.e., an ectodomain antigen of the introduced protein associated with the disease) within 2 days, 5 days, 10 days, 15 days, 20 days, or, preferably , 30 days after the first administration and prior to any subsequent administration. The immune response can be determined in an individual or in an animal model, any response then being correlated or extrapolated to a predicted response in the individual, for example, a human. In one embodiment, the present invention provides methods for preventing an NDV infection and/or infections by another infectious agent other than NDV in a bird, the method comprising administering a first dose of an immunogenic formulation comprising a chimeric NDV of the invention. , any chimeric NDV comprises a fusion protein containing a heterologous protein sequence, to said individual of an effective amount of the chimeric virus of the invention, wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml , 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or greater of antibodies that immunospecifically bind to a chimeric virus antigen and/or antibodies that immunospecifically bind to a fusion protein antigen 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after the first administration and prior to any subsequent administration. In some embodiments, the dose of chimeric influenza virus administered to the subject or animal model is 102.5 x 102, 103.5 x 10, 10.5 x 104, 105.5 x 105, 106.5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu.

[0182] In one embodiment, the present invention provides methods for treating at least one disease (e.g., an NDV infection and/or infections by another infectious agent that is not NDV) in an individual, the methods comprising administering to the said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric NDV of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg /ml or greater of antibodies that immunospecifically bind to an antigen or epitope of the chimeric virus backbone 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after the first administration and prior to any administration subsequent. In another embodiment, the present invention provides methods for treating at least one disease (e.g., an NDV infection and/or infections by another infectious agent other than NDV) in an individual, the methods comprising administering to said individual a first dose of an effective amount of an immunogenic formulation comprising chimeric NDV of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or of antibodies that bind immunospecifically to a fusion protein antigen (i.e., an ectodomain antigen of the introduced protein associated with the disease) in 2 days, 5 days, 10 days, 15 days, 20 days, or, preferably, 30 days after the first administration and before any subsequent administration. The immune response can be determined in an individual or in an animal model, any response then being correlated or extrapolated to a predicted response in the individual, for example, a human. In one embodiment, the present invention provides methods for treating an NDV infection and/or infections with another infectious agent other than NDV in a bird, the method comprising administering a first dose of an immunogenic formulation comprising a chimeric NDV of the invention. , any chimeric NDV comprises a fusion protein containing a heterologous protein sequence, to said individual of an effective amount of the chimeric virus of the invention, wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml , 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or greater of antibodies that immunospecifically bind to a chimeric virus antigen and/or antibodies that immunospecifically bind to a fusion protein antigen 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after the first administration and prior to any subsequent administration. In some embodiments, the dose of chimeric influenza virus administered to the subject or animal model is 102.5 x 102, 103.5 x 103, 104.5 x 104, 105.5 x 105, 106.5 x 106, 107, 5 x 107, 10s, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu.

[0183] In one embodiment, the present invention provides methods for controlling and/or ameliorating at least one disease (e.g., an NDV infection and/or infections by another infectious agent other than NDV) in an individual, the methods comprising administering to said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric NDV of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the amount effective is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg /ml, 100 µg/ml or greater of antibodies that immunospecifically bind to an antigen or epitope on the backbone of the chimeric virus 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after the first administration and prior to any subsequent administration. In another embodiment, the present invention provides methods for controlling and/or ameliorating at least one disease (e.g., an NDVe infection/or infections with another infectious agent other than NDV) in an individual, the methods comprising administering to the said individual a first dose of an effective amount of an immunogenic formulation comprising a chimeric NDV of the invention, which chimeric virus comprises a fusion protein of a heterologous sequence (e.g., a disease antigen), wherein the effective amount is the amount which results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or greater of antibodies that immunospecifically bind to a fusion protein antigen (i.e., an ectodomain antigen of the introduced protein associated with the disease) within 2 days, 5 days, 10 days, 15 days, 20 days, or preferably 30 days after the first administration and prior to any subsequent administration. The immune response can be determined in an individual or in an animal model, any response then being correlated or extrapolated to a predicted response in the individual, for example, a human. In one embodiment, the present invention provides methods for controlling and/or ameliorating an NDV infection and/or infections by another infectious agent other than NDV in a bird, the method comprising administering a first dose of an immunogenic formulation comprising an chimeric NDV of the invention, any chimeric NDV comprises a fusion protein containing a heterologous protein sequence, to said individual of an effective amount of the chimeric virus of the invention, wherein the effective amount is the amount that results in a serum titer of about 10 µg/ml, 20 µg/ml, 30 µg/ml, 40 µg/ml, 50 µg/ml, 60 µg/ml, 70 µg/ml, 80 µg/ml, 100 µg/ml or greater of antibodies that bind immunospecifically to a chimeric virus antigen and/or antibodies that bind immunospecifically to a fusion protein antigen 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days

[0184] The present invention further provides methods for preventing, treating and/or controlling at least one disease, the methods comprising administering to said individual an effective amount of an immunogenic formulation comprising a chimeric influenza virus of the invention, wherein the effective amount is the amount that results in a reduction in mortality, a reduction in hospitalization, a reduction in the severity of the disease and/or a reduction in the clinical symptoms of the disease relative to the individual not administered with the immunogenic formulation of the invention. In some embodiments the individual is a human. In some embodiments, the dose of chimeric influenza virus administered to the subject is 102.5 x 102, 103.5 x 103, 104.5 x 104, 105.5 x 105, 106.5 x 106, 107.5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu.

[0185] In another embodiment, the present invention provides methods for preventing, treating and/or controlling at least one disease (e.g., an avian influenza infection and/or infection by another infectious agent other than avian influenza) in an individual (preferably bird), the methods comprising administering to said individual an effective amount of an immunogenic formulation comprising a chimeric avian influenza virus of the invention, wherein the effective amount is the amount that results in a reduction in the titer or number of agents infectious diseases, reduction in mortality, reduction in hospitalization, reduction in severity of infection and/or reduction in clinical symptoms of infection relative to an individual not administered with the immunogenic formulation of the invention. In some embodiments, the dose of chimeric avian influenza virus administered to the subject is 102.5 x 102, 103.5 x 103, 104.5 x 104, 105.5 x 105, 106.5 x 106, 107, 5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu. In some embodiments, administration of an immunogenic formulation results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or greater reduction in replication of the infectious agent relative to a subject not administered with the immunogenic formulation of the invention as determined within 2 days, 5 days, 10 days, 15 days, 20 days or, Preferably, 30 days after said administration by any method known in the art or exemplified herein (e.g., determination of viral title). In other embodiments, administration of an immunogenic formulation of the invention results in a 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, or 100-fold reduction in the replication of the infectious agent or the infectious agent load relative to an individual not administered an immunogenic formulation of the invention as determined at 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after said administration by any method known in the art or exemplified herein (e.g. determination of viral titer or bacterial load and/or concentration).

[0186] In another embodiment, the present invention provides methods for preventing, treating and/or ameliorating at least one disease (e.g., an NDV infection and/or infection with another infectious agent other than NDV) in an individual (e.g., a bird), the methods comprising administering to said individual an effective amount of an immunogenic formulation comprising an NDV chimeric virus of the invention, wherein the effective amount is the amount that results in a reduction in the titer or number of agents infectious diseases, reduction in mortality, reduction in hospitalization, reduction in severity of infection and/or reduction in clinical symptoms of infection relative to an individual not administered with the immunogenic formulation of the invention. In some embodiments, the dose of the NDV chimeric virus administered to the subject is 102.5 x 102, 103.5 x 103, 104.5 x 104, 105.5 x 105, 106.5 x 106, 107.5 x 107, 108, 5 x 108, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011 or 1012 pfu. In some embodiments, administration of an immunogenic formulation results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% or greater reduction in replication of the infectious agent relative to a subject not administered with the immunogenic formulation of the invention as determined within 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after said administration by any method known in the art or exemplified herein (e.g., determination of viral title). In other embodiments, administration of an immunogenic formulation results in a 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 75, or 100-fold reduction in the replication of the infectious agent or the burden of infectious agent relative to an individual not administered with the immunogenic formulation of the invention as determined within 2 days, 5 days, 10 days, 15 days, 20 days or, preferably, 30 days after said administration by any method known in the art or exemplified herein ( e.g. viral titer determination).

[0187] The amount of the immunogenic formulation of the invention that will be effective in treating, preventing and/or ameliorating a particular disease (e.g., viral infection) will depend on the nature of the disease, and can be determined by standard clinical techniques. In addition, in vitro testing can optionally be employed to help identify optimal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration, and severity of the infection or disorder, and must be decided according to the clinician's judgment and the circumstances of each individual. However, appropriate dosage ranges for administration are generally about 102.5x102, 103.5x103, 104.5x104, 105.5x105, 106.5x106, 107.5x107, 108. 5 x 10s, 1 x 109, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011. 5 x 1011 or 1012 pfu, and more preferably about 104 to about 1012, and may be administered to an individual one , two, three or more times with breaks as many times as necessary. Effective doses can be extrapolated from dose response curves derived from in vitro test systems or animal models.

[0188] In various embodiments, the immunogenic formulations of the invention or antibodies generated by the chimeric viruses of the invention are administered to an individual in combination with one or more therapies (e.g., antiviral or immunomodulatory therapies) for the prevention of at least one illness (e.g., an influenza infection and/or infection with another infectious agent that is not an influenza virus). In other embodiments, the immunogenic formulations of the invention or antibodies generated by the chimeric viruses of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the treatment of at least one disease ( for example, an influenza infection and/or infection with another infectious agent that is not an influenza virus). In still other embodiments, the immunogenic formulations of the invention or antibodies generated by the chimeric viruses of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the control and/or improvement of at least one disease (e.g., an influenza infection and/or infection with another infectious agent other than influenza viruses). In a specific embodiment, the immunogenic formulations of the invention or antibodies generated by the chimeric viruses of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the prevention of an influenza infection. avian influenza and/or infection with another infectious agent other than avian influenza viruses. In another specific embodiment, the immunogenic formulations of the invention or antibodies generated by the chimeric viruses of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the treatment of an influenza infection. avian influenza and/or infection with another infectious agent other than avian influenza viruses. In still other embodiments, immunogenic formulations of the invention or antibodies generated by the chimeric viruses of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for the prevention of an NDV infection. and/or infection with an infectious agent other than NDV. In still other embodiments, immunogenic formulations of the invention or antibodies generated by the chimeric viruses of the invention are administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies) for treating an NDV infection. and/or infection with an infectious agent other than NDV. In some embodiments, therapies (e.g., prophylactic and therapeutic agents) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about from 5 hours to about 6 hours apart, from about 6 hours to about 7 hours apart, from about 7 hours to about 8 hours apart, from about 8 hours to about 9 hours apart, in about 9 hours to about 10 hours apart, in about 10 hours to about 11 hours apart, in about 11 hours to about 12 hours apart, in about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. In preferred embodiments, two or more therapies are administered in the same patent visit.

[0189] Any antiviral agent well known to those skilled in the art can be used in the formulations (e.g., vaccine formulations) and methods of the invention. Non-limiting examples of antiviral agents include proteins, polypeptides, peptides, fusion protein antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and/or reduce the attachment of a virus to its receptor, internalization of a virus in a cell, replication of a virus, or release of viruses from a cell. In particular, antiviral agents include, but are not limited to, nucleoside analogues (e.g., zidovudine, acyclovir, ganciclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, alpha- interferons and other interferons, and AZT.

[0190] In specific embodiments, the antiviral agent is an immunomodulatory agent that is immunospecific for a viral antigen. As used herein, the term "viral antigen" includes, but is not limited to, any viral peptide, polypeptide and protein (e.g., HIV gp120, HIV nef, RSV glycoprotein F, RSV glycoprotein G, influenza virus neuraminidase , influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB, gC, gD, and gE), and hepatitis B surface antigen) that is capable of eliciting an immune response. Antibodies usable in this invention to treat a viral infectious disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including by way of example and not by way of limitation: adenovirdiae (e.g., mastadenoviruses and aviadenoviruses), herpesviridae (e.g., herpes viruses simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g., levivirus, MS2 stage enterobacteria, allolevirus), poxviridae (e.g., cordopoxvirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporiipoxviruses, suipoxviruses, molluscipoxviruses, and entomopoxvirinae), papovaviridae (e.g., polyomaviruses and papillomaviruses), paramyxoviridae (e.g., paramyxoviruses, influenza 1 paraviruses, mobilliviruses (e.g., measles virus), rubulaviruses (e.g., mumps), pneumonovirinae (e.g., pneumovirus, human respiratory syncytial virus), and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus)), picornaviridae (e.g., enterovirus, rhinovirus, hepatovirus (e.g., hepatitis A virus) human), cardioviruses, and apthoviruses), reoviridae (e.g., orthoreovirus, orbivirus, rotavirus, cypovirus, fijivirus, phytovirus, and oryzavirus), retroviridae (e.g., mammalian type B retrovirus, mammalian type C retrovirus, avian C, type D retrovirus strain, BLV-HTLV retrovirus, lentivirus (e.g., human immunodeficiency virus 1 and human immunodeficiency virus 2), spumavirus), flaviviridae (e.g., hepatitis C virus, dengue virus, West Nile), hepadnaviridae (e.g. hepatitis B virus), togaviridae (e.g. alphaviruses (e.g. sindbis virus) and rubiviruses (e.g. rubella virus)), rhabdoviridae (e.g. vesiculovirus, lyssavirus, ephemerovirus , cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, Lymphocytic Choriomeningitis virus, Ippy virus, and lassavirus), and coronaviridae (e.g., coronavirus and torovirus).

[0191] Antibacterial agents and therapies well known to those skilled in the art for the prevention, treatment, control, or amelioration of bacterial infections can be used in compositions (e.g., immunogenic formulations) and methods of the invention. Non-limiting examples of antibacterial agents include proteins, polypeptides, peptides, protein fusions, antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit or reduce a bacterial infection, inhibit or reduce bacterial replication, or inhibit or reduce the spread of bacteria to other individuals. In particular, examples of antibacterial agents include, but are not limited to, penicillin, cephalosporin, imipenem, aztreonam, vancomycin, cycloserine, bacitracin, chloramphenicol, erythromycin, clindamycin, tetracycline, streptomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, spectinomycin , trimethoprim, norfloxacin, rifampin, polymyxin, amphotericin B, nystatin, quetoconazole, isoniazid, metronidazole, and pentamidine. Antibacterial therapies and their dosages, routes of administration and recommended uses are known in the art and have been described in the literature as the Physician's Desk Reference (56th ed., 2002). Additional information on respiratory infections and antibacterial therapies is available in the Cecil Textbook of Medicine (18th ed., 1988).

[0192] Antifungal agents and therapies well known to those skilled in the art for preventing, controlling, treating, and/or ameliorating a fungal infection or one or more symptoms thereof (e.g., a fungal respiratory infection) can be used in the compositions ( e.g., immunogenic formulations) and methods of the invention. Non-limiting examples of antifungal agents include proteins, polypeptides, peptides, fusion proteins, antibodies, nucleic acid molecules, organic molecules, inorganic molecules, and small molecules that inhibit and/or reduce fungal infection, inhibit and/or reduce replication of fungus, or inhibit and/or reduce the spread of fungi to other individuals. Specific examples of antifungal agents include, but are not limited to, azole-type drugs (e.g., miconazole, quetoconazole (NIZORAL®), caspofungin acetate (CANCID AS®), imidazole, triazoles (e.g., fluconazole (DIFLUCAN®)) ), and itraconazole (SPORANOX®)), polyene (e.g., nystatin, amphotericin B (FUNGIZONE®) amphotericin B lipid complex ("ABLC)(ABELCET®), amphotericin B colloidal dispersion ("ABCD")(AMPHOTEC® ), liposomal amphotericin B (AMBISONE®)), potassium iodide (KI), pymiridine (e.g., flucytocin (ANCOBON®)), and voriconazole (VFEND®). Antifungal therapies and their dosages, routes of administration, and recommended uses are known in the art and have been described in the literature as in Dodds et al, 2000 Pharmacotherapy 20(11) 1335-1355, the Physician's Desk Reference (57th ed. 2003) and the Merk Manual of Diagnosis and Therapy (17th ed. ., 1999).

[0193] In some embodiments, an immunogenic formulation of the invention is administered to an individual as a single dose followed by a second dose 3 to 6 weeks later. According to these embodiments, booster inoculations may be administered to the subject at intervals of 6 to 12 months following the second inoculation. In one embodiment, the subject is a mammal. In another embodiment, the individual is a bird. In yet another embodiment the individual is a human. In a more preferred embodiment, the subject is a chicken at risk of contracting an NDV or avian influenza virus infection.

[0194] In some embodiments, the administration of the same immunogenic formulations of the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

5.6 Biological Tests 5.6.1 In Vitro Tests

[0195] Growth of the chimeric viruses of the present invention can be evaluated by any method known in the art or described herein (e.g., in cell culture (e.g., chicken embryonic kidney cell cultures or chicken embryonic fibroblast (CEF) cultures) ). Growth of the attenuated chimeric viruses of the invention can be evaluated in both IFN-competent and IFN-deficient cells. In a specific embodiment, CEF cells are infected at an MOI of 0.0005 and 0.001; 0.001 and 0.01; 0 .01 and 0.1; 0.1 and 1, or 1 and 10; or an MOI of 0.0005; 0.001; 0.005; 0.01; 0.05; 0.1; 0.5; 1; 5 or 10 and incubated with serum-free media supplemented with 5% allantoic fluid. Viral titers are determined in the supernatant by HA plated onto CEF cells as described below. Other cells in which viral titers can be assessed include, but are not limited to, cells EFK-2, Vero cells, human primary umbilical vein endothelial cells (HUVEC), human epithelial cell lines H292 and HeLa cells.

[0196] Incorporation of the fusion protein into the virion of the chimeric viruses of the present invention can be evaluated by any method known in the art or described herein (for example, in cell culture, animal model or viral culture in embryonated eggs). For example, cell culture viral particles from allantoic fluid of embryonated eggs can be purified by centrifugation through a sucrose buffer and subsequently analyzed for fusion protein expression by Western blotting using methods well known in the art.

[0197] Viral tests include those that measure altered viral replication (as determined, for example, by plaque formation) or the production of viral proteins (as determined, for example, by Western Blot analysis) or viral RNA (as determined, for example, by RT-PCR or Northern Blot analysis) on cells cultured in vitro using methods that are well known in the art.

[0198] Antibodies generated by the chimeric viruses of the present invention or fragments thereof can be characterized in a variety of ways well known to one skilled in the art (e.g., ELISA, Surface Plasmon Resonance Display (BIAcore) tests, Western blot, immunofluorescence, immunostaining and/or microneutralization). In particular, antibodies generated by the chimeric viruses of the present invention or fragments thereof can be tested for the ability to immunospecifically bind to a backbone chimeric virus antigen or a fusion protein antigen or epitope. Such a test can be performed in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), on beads (Lam, 1991, Nature 354:82-84), on chips (Fodor, 1993, Nature 364: 555-556), in bacteria (U.S. Patent Nos. 5,223,409), in spores (U.S. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), in plasmids (Cull et al, 1992, Proc. Natl . Acad. Sci. USA 89:1865-1869) or in phages (Scott and Smith, 1990, Science 249:386-390; Cwirla et al, 1990, Proc. Natl. Acad. Sci. USA 87:63786382; and Felici , 1991, J. MoI. Biol. 222:301-310) (each such reference is incorporated herein in its entirety by reference). Antibodies generated by the chimeric viruses of the present invention or fragments thereof that have been identified to immunospecifically bind to a backbone chimeric virus antigen or a fusion protein antigen or epitope can then be tested for their specificity for said antigen.

[0199] Antibodies generated by the chimeric viruses of the present invention or fragments thereof can be tested for immunospecific binding to an antigen of the chimeric virus of the invention (for example, an antigen or epitope from the chimeric virus backbone or an antigen or epitope from the fusion protein (e.g., an antigen associated with a disease)) and cross-reactivity with other antigens by any method known in the art. Immunotests that can be used to analyze immunospecific binding and cross-reactivity include, but are not limited to, competitive and non-competitive testing systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme-linked immunosorbent assay), sandwich immunoassays. ", immunoprecipitation tests, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion tests, agglutination tests, complement fixation tests, immunoradiometric tests, fluorescent immunotests, protein A immunotests, to name just a few examples. Such tests are routine and well known in the art (see, e.g., Ausubel et al, eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference here in its entirety). Exemplary immunotests are described briefly below (but are not intended to be limited).

[0200] Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the interest to the cell lysate, incubating for a period of time (e.g., 1 to 4 hours) at 40°C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or so. further at 40°C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, for example, Western Blot analysis. One skilled in the art should have knowledge of the parameters that can be modified to increase antibody binding to an antigen and decrease preceding steps (e.g., pre-clearing the cell lysate with sepharose beads). For a further discussion of immunoprecipitation protocols see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

[0201] Western blot analysis generally comprises preparation of protein samples, electrophoresis of the protein samples on a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the sample to protein from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, incubating the membrane in blocking solution (e.g., PBS with 3% BSA or skim milk), washing the membrane in a wash buffer (e.g., PBS -Tween 20), incubating the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in wash buffer, incubating the membrane with a secondary antibody (which recognizes the primary antibody, e.g. a anti-human antibody) conjugated to an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One skilled in the art should have knowledge of the parameters that can be modified to increase the detected signal and reduce background noise. For another discussion regarding Western Blot protocols see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

[0202] ELISAs comprise preparing antigen by coating the well of a 96-well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase). into the cavity and incubating for a period of time, and detecting the presence of the antigen. In ELISAs, the antibody of interest does not need to be conjugated to a detectable compound; instead, a secondary antibody (which recognizes the antibody of interest) conjugated to a detectable compound can be added to the well. Furthermore, instead of coating the well with the antigen, the antibody can be coated with the well. In this case, a second antibody conjugated to a detectable compound can be added following the addition of the antigen of interest to the coated well. One skilled in the art should have knowledge of parameters that can be modified to increase the detected signal as well as other variations of ELISAs known in the art. In a preferred embodiment, an ELISA can be performed by coating a high-binding 96-well microtiter plate (Costar) with 2µg/ml rhu-IL-9 in PBS overnight. Following three washes with PBS, the plate is incubated with three-fold serial dilutions of Fab at 25°C for 1 hour. Following another three washes of PBS, 1 µg/ml conjugated alkaline anti-human kappa phosphatase is added and the plate is incubated for 1 hour at 25 oC. Following the three washes with PBST, phosphatase activity is determined in 50µl/AMP/PPMP substrate. Reactions are stopped and absorbance at 560 nm is determined with a VMAX microplate reader. For further discussion regarding ELISA see, for example, Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc, New York at 11.2.1.

[0203] The binding affinity of an antibody to an antigen and the dissociation rate of an antibody-antigen interaction can be determined by competitive binding tests. An example of a competitive binding test is a radioimmunotest comprising incubating labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and detecting the antibody bound to the labeled antigen. The affinity of the antibody of the present invention or a fragment thereof to an IL-9 polypeptide and the binding dissociation rates can be determined from the data by Scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, an IL-9 polypeptide is incubated with an antibody of the present invention conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled secondary antibody.

[0204] In a preferred embodiment, BIAcore kinetic analysis is used to determine the rates of "associated" and "disassociated" binding of antibodies of the invention to a chimeric virus antigen of the invention (e.g., an antigen or epitope of the chimeric virus backbone or an antigen or epitope of a fusion protein (e.g., an antigen associated with a disease)). BIAcore kinetic analysis comprises analyzing the binding and dissociation of polypeptide comprising the antigen of interest from the chips with immobilized antibody generated by the chimeric viruses of the invention on their surface. A typical BIAcore kinetic study involves injecting 250 µL of an antibody reagent (mAb, Fab) at varying concentration in HBS buffer containing 0.005% Tween-20 onto a sensor chip surface to which the antigen has been immobilized. The flow rate is kept constant at 75 µL/min. Dissociation data is collected in 15 min or longer as needed. Following each injection/dissociation cycle, the bound mAb is removed from the antigen surface using brief 1 min pulses of dilute acid, typically 10-100 mM HCl, although other regenerants are employed as circumstances warrant. More specifically, to measure the rates of association, kon, and dissociation, koff, the polypeptide comprising the antigen is directly immobilized on the sensor chip surface through the use of standard amine bond chemistries, i.e. the EDC/NHS Method ( EDC= N-diethylaminopropyl)-carbodiimide). Briefly, a solution of 5-100 nM of the polypeptide comprising the antigen in 10mM NaOAc, pH4 or pH5 is prepared and passed onto the activated EDC/NHS surface until approximately 30-50 RU's of antigen is immobilized. Following this, the unreacted active esters are "terminated" with an injection of 1M Et-NH2. A blank surface, containing no antigen, is prepared under identical immobilization conditions for reference purposes. Once an appropriate surface has been prepared, appropriate dilution series of each of the antibody reagents are prepared in HBS/Tween-20, and passed under both the antigen and reference cell surface, which are connected in series. The concentration rates of antibodies that are prepared vary depending on what the binding equilibrium constant, KD, is estimated to be. As described above, bound antibody is removed after each injection/dissociation cycle using an appropriate regenerant.

[0205] Antibodies generated by the chimeric viruses of the invention or fragments thereof can also be tested for their ability to inhibit the binding of an antigen of the chimeric virus of the invention (for example, an antigen or epitope from the chimeric virus backbone or a fusion protein antigen or epitope (e.g., an antigen associated with a disease)) to a host cell receptor using techniques known to those skilled in the art. For example, receptors expressing cells known to bind said antigens may be contacted with antigen in the presence or absence of an antibody generated by the chimeric virus of the invention or fragment thereof and the ability of the antibody or fragment thereof to inhibit antigen binding may be determined. measured by, for example, flow cytometry or a scintillation test. The antigen or antibody or antibody fragment may be labeled with a detectable compound such as a radioactive label (e.g., 32P, 35S, and 1251) or a fluorescent label (e.g., fluorescin isothiocyanate, rhodamine, phycoerythin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine) in order to allow detection of an interaction between the antigen and a cellular receptor. Alternatively, the ability of antibodies generated by the chimeric viruses of the invention or fragments thereof to inhibit an antigen of the chimeric virus of the invention (e.g., a chimeric virus backbone antigen or epitope or a fusion protein antigen or epitope (e.g., For example, an antigen associated with a disease)) binding to a receptor can be determined in cell-free assays. For example, a polypeptide comprising the antigen can be contacted with an antibody or fragment thereof and the ability of the antibody or antibody fragment to inhibit the polypeptide from binding to a cellular receptor can be determined. Preferably, the antibody or antibody fragment is immobilized on a solid support and the polypeptide is labeled with a detectable compound. Alternatively, a polypeptide comprising the antigen is immobilized on a solid support and the antibody or fragment thereof is labeled with a detectable compound.

5.6.2 In Vivo Tests

[0206] The virulence of the chimeric viruses of the present invention can be evaluated in an individual, in particular birds, or in an animal model thereof. In one example, the ability to induce lung injury and cause infection in an animal model of virus infection is compared for wild-type viruses and placebo viruses. Lung lesions can be assessed as the percentage of lung lobes that are healthy by visual inspection. Animals are sacrificed 5 days p.i. by intravenous administration of pentobarbital, and his lungs are removed in toto. The percentage of the surface of each lung lobe that is affected by macroscopic lesions is estimated visually. The percentages are calculated to obtain an average value for the 7 lung lobes of each animal. In other tests, nasal swabs may be tested to determine viral load or titer. Nasal swabs may be taken during necropsy to determine viral load post infection.

[0207] For quantification of viruses in tissue samples, tissue samples are homogenized in phosphate-buffered saline (PBS), and dilutions of clarified homogenates adsorbed for 1 h at 37°C onto cell monolayers (e.g., CEF or MDCK). Infected monolayers are then overlaid with a minimal essential culture medium solution containing 0.1% bovine serum albumin (BSA), 0.01% DEAE-dextran, 0.1% NaHCO3, and 1% agar. Plates are incubated 2 to 3 days until the plates can be visualized. Tissue culture infectious dose (TCID) tests to titrate viruses from infected samples

[0208] In still other tests, histopathological evaluations are performed after infection. Nasal turbinates and trachea can be examined for epithelial changes and subepithelial inflammation. The lungs can be examined for bronchiolar epithelial changes and peribronchiolar inflammation in large, medium, and small or terminal bronchioles. The alveoli are also evaluated for inflammatory changes. Medium bronchioles are graded on a scale of 0 to 3+ as follows: 0 (normal: lined by medium to tall columnar epithelial cells with ciliated apical borders and basal pseudostratified nuclei; minimal inflammation); 1+ (regular columnar epithelial layer with only slightly increased proliferation; cilia still visible in many cells); 2+ (prominent changes in the epithelial layer ranging from attenuated to enhanced proliferation; disorganized cells and irregular layering at the luminal border); 3+ (epithelial layer markedly disrupted and disorganized with necrotic cells visible in the lumen; some bronchioles attenuated and others in marked reactive proliferation).

[0209] The trachea is graded on a scale from 0 to 2.5+ as follows: 0 (normal: lined by medium to tall columnar epithelial cells with ciliated apical border, basal and pseudostratified nuclei. Cytoplasm evident between apical borders and nuclei Occasional small focus with squamous cells); 1+ (focal squamous metaplasia of the epithelial layer); 2+ (diffuse squamous metaplasia of much of the epithelial layers, cilia may be focally evident); 2.5+ (diffuse squamous metaplasia with very few cilia evident).

[0210] Virus immunohistochemistry is performed using a viral-specific monoclonal antibody (e.g., NP, N, or HN-specific monoclonal antibodies). Staining is graded from 0 to 3+ as follows: 0 (uninfected cells); 0.5+ (some infected cells); 1+ (a few infected cells, such as widely separated individual cells); 1.5+ (some infected cells, such as single cells separated widely and in small clusters); 2+ (moderate number of infected cells, usually affecting clusters of adjacent cells in parts of the bronchioles lining the epithelial layer, or in small sublobular foci in alveoli); 3+ (numerous infected cells, affecting most of the epithelial layer in bronchioles, or widespread in large sublobular foci in alveoli).

5.6.3 Determination of viral titer

[0211] Viral titer is determined by inoculating serial dilutions of chimeric virus into cell cultures (e.g., CEF or MDCK), chicken embryos, or live animals (e.g., birds). After incubating the virus for a specific time, the virus is isolated using standard methods.

[0212] The HA test can be performed in 96-well V-bottom plates. Double serial dilutions of each sample in PBS are incubated for 1 h on ice with an equal volume of a 0.5% erythrocyte suspension of chicken in PBS. Positive cavities contain an adherent and homogeneous layer of erythrocytes; negative cavities contain a non-adherent bead.

[0213] Physical quantification of viral titer can be performed using PCR applied to viral supernatants (Quinn & Trevor, 1997; Morgan et al, 1990), hemagglutination tests, tissue culture infectious doses (TCID50) or egg infectious doses ( EID50).

5.6.4 Toxicity studies

[0214] The toxicity and/or efficacy of the compositions (e.g., immunogenic formulations) of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the lethal dose for 50% of population) and ED50 (the therapeutically effective dose in 50% of the population). The dose relationship between toxic and therapeutic effects is the therapeutic index and this can be expressed as the LD50/ED50 ratio. Therapies that exhibit large therapeutic indices are

[0215] Data obtained from cell culture tests and animal studies can be used in formulating a dosage range of therapies for use in individuals. The dosage of such agents is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary in this range depending on the dosage form employed and route of administration used. For any therapy used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture tests. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a maximal partial inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in individuals (e.g., horses). Levels in plasma can be measured, for example, by high-performance liquid chromatography.

[0216] Furthermore, any tests known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic utility of a composition (e.g., vaccine formulation), a combination therapy described herein for viral infection or a condition or symptoms associated therewith, an infection other than a viral infection or a condition or symptom associated therewith, or a condition in which a chimeric attenuated virus of the invention is used as a vector to induce an immune response to an antigen associated with the same. condition.

5.7 Specific embodiments of the invention

[0217] The present invention provides a chimeric avian influenza virus, comprising a fusion protein, having

[0218] (i) an ectodomain comprising a heterologous peptide sequence, which heterologous sequence comprises at least one epitope of a

[0219] (ii) a cytoplasmic and transmembrane domain of a glycoprotein encoded by an essential gene of an influenza virus, wherein the fusion protein is incorporated into an avian influenza virus, wherein the function of the essential gene is supplied by the fusion protein or glycoprotein native to the avian influenza virus. In some embodiments, the essential gene of an influenza virus is a hemagglutinin (HA) gene. In other embodiments, the essential gene of an influenza virus is a neuraminidase (NA) gene. In some embodiments, the chimeric avian influenza virus is attenuated. According to these embodiments, the chimeric avian influenza virus can be attenuated by mutations in the NS1 gene.

[0220] The present invention provides a chimeric avian influenza virus, comprising a fusion protein, having

[0221] (i) an ectodomain of an NDV HN protein fused to

[0222] (ii) a cytoplasmic and transmembrane domain of an influenza virus NA protein, wherein the fusion protein is incorporated into an avian influenza virus, wherein the function of the NA protein is supplied by the fusion protein or by the glycoprotein native to the avian influenza virus. In some embodiments, the chimeric avian influenza virus is attenuated. According to these embodiments, the chimeric avian influenza virus can be attenuated by mutations in the NS1 gene.

[0223] The present invention provides an attenuated chimeric influenza virus, comprising a fusion protein, having

[0224] (i) an ectodomain comprising a heterologous peptide sequence, which heterologous sequence comprises at least one epitope of a protective antigen of an infectious agent, other than influenza, or of an antigen associated with a disease of a protective antigen of a infectious agent, other than influenza fused to

[0225] (ii) cytoplasmic domain of a transmembrane and a glycoprotein encoded by an essential gene of an influenza virus, in which the fusion protein is incorporated into an attenuated influenza virus, in which the function of the essential gene is supplied by the fusion protein or glycoprotein native to the attenuated influenza virus. In some embodiments, the essential gene of an influenza virus is a hemagglutinin (HA) gene. In other embodiments, the essential gene of an influenza virus is a neuraminidase (NA) gene.

[0226] The present invention provides a chimeric NDV, comprising a fusion protein, having

[0227] (i) an ectodomain comprising a heterologous peptide sequence, which heterologous sequence comprises at least one epitope of a protective antigen of an infectious agent, other than NDV, or of an antigen associated with a disease fused to

[0228] (ii) a cytoplasmic and transmembrane domain of a glycoprotein encoded by an essential gene of an NDV,

[0229] wherein the fusion protein is incorporated into an NDV, wherein the essential gene function is supplied by the fusion protein or by the glycoprotein native to the NDV.

[0230] The present invention provides a chimeric avian influenza virus, comprising an enveloped influenza virus NA segment encoding a neuraminidase fusion protein, wherein the NA open reading frame is modified such that the nucleotides encoding the ectodomain of NA are replaced by nucleotides encoding an ectodomain of a neuraminidase antigen from an infectious agent other than influenza that is anchored by the N-terminus, so that neuraminidase fusion protein is expressed and incorporated into the chimeric avian influenza virus.

[0231] The present invention provides a chimeric avian influenza virus, comprising an enveloped influenza virus HA segment encoding the hemagglutinin fusion protein, wherein the HA open reading frame is modified such that the nucleotides encoding the HA ectodomain are replaced by nucleotides encoding an ectodomain of an HA antigen.

[0232] so that the hemagglutinin fusion protein is expressed and incorporated into the chimeric avian influenza virus.

[0233] The present invention provides a chimeric avian influenza virus, comprising a bicistronic enveloped influenza virus HA segment, comprising: (a) a first open reading frame encoding an avian influenza hemagglutinin protein, and (b ) a second open reading frame encoding a hemagglutinin fusion protein, wherein the nucleotides encoding the hemagglutinin ectodomain are replaced by nucleotides encoding a heterologous peptide sequence, which heterologous sequence comprises at least one epitope of a protective antigen of a infectious agent, other than influenza, or an antigen associated with a disease that is anchored by the C-terminus, such that both the influenza hemagglutinin and the fusion protein are expressed and incorporated into the chimeric avian influenza virus.

[0234] The present invention provides a chimeric avian influenza virus, comprising a bicistronic enveloped influenza virus NA segment, comprising: (a) a first open reading frame encoding an avian influenza neuraminidase protein, and (b ) a second open reading frame encoding a neuraminidase fusion protein, wherein the nucleotides encoding the neuraminidase ectodomain are replaced by nucleotides encoding the heterologous peptide sequence, which heterologous sequence comprises at least one epitope of a protective antigen of a infectious agent, other than influenza, or an antigen associated with a disease that is anchored by the N-terminus, such that both the influenza neuraminidase and the fusion protein are expressed and incorporated into the chimeric avian influenza virus.

[0235] The present invention provides a chimeric avian influenza virus, comprising an enveloped influenza virus NA segment encoding a neuraminidase fusion protein, wherein the NA open reading frame is modified such that the nucleotides encoding the ectodomain of NA are replaced by nucleotides encoding an ectodomain of an NDV HN antigen,

[0236] so that the neuraminidase fusion protein is expressed and incorporated into the chimeric avian influenza virus.

[0237] In some embodiments, the chimeric avian influenza virus of paragraphs 209-211 and 213-217 comprising an enveloped NS1 gene segment encodes the modified NS1 protein that reduces the cellular interferon antagonist activity of the virus. In other embodiments, the chimeric avian influenza virus of pages 209-211 and 213-217 comprises an HA segment having an open reading frame modified to remove the hemagglutinin polybasic cleavage site. In still other embodiments, the chimeric avian influenza virus of paragraph 215, in which the first open reading frame is modified to remove the hemagglutinin polybasic cleavage site.

[0238] The present invention provides a recombinant nucleic acid molecule (e.g., recombinant DNA molecules) encoding the NA segment of paragraphs 213 and 216. The present invention further provides recombinant nucleic acid molecules (e.g., DNA molecules recombinant) encoding the HA segment of paragraphs 214-215.

[0239] The present invention provides methods for propagating the chimeric avian influenza viruses of paragraphs 209-211 and 213-218, comprising cultivating the chimeric avian influenza viruses in an embryonated egg or a cell line that is susceptible to infection by avian influenza viruses. avian influenza. The present invention further provides methods for producing an immunogenic formulation, the method comprising: (a) propagating the chimeric avian influenza virus of paragraphs 209-211 and 213-218 in an embryonated egg or cell line that is susceptible to virus infection from avian influenza; and (b) collecting the progeny virus, wherein the virus is grown in sufficient quantities and under sufficient conditions that the virus is free from contamination such that the progeny virus is suitable for use in an immunogenic formulation, e.g. vaccine formulation.

[0240] The present invention provides an attenuated chimeric influenza virus comprising an enveloped influenza virus NA segment encoding the neuraminidase fusion protein, wherein the NA open reading frame is modified such that the nucleotides encoding the ectodomain of NA are replaced by nucleotides encoding an ectodomain of a neuraminidase antigen from an infectious agent other than influenza that is anchored by the N-terminus, such that the neuraminidase fusion protein is expressed and incorporated into the attenuated chimeric avian influenza virus .

[0241] The present invention provides an attenuated chimeric influenza virus comprising an enveloped influenza virus HA segment encoding a hemagglutinin fusion protein, wherein the HA open reading frame is modified such that the nucleotides encoding the ectodomain of HA are replaced by nucleotides encoding an ectodomain of a hemagglutinin antigen from an infectious agent other than influenza that is anchored by the C-terminus, so that the hemagglutinin fusion protein is expressed and incorporated into the attenuated chimeric influenza virus.

[0242] The present invention provides an attenuated chimeric avian influenza virus, comprising a bicistronic enveloped influenza virus HA segment, comprising: (a) a first open reading frame encoding an avian influenza hemagglutinin protein, and ( b) a second open reading frame encoding a hemagglutinin fusion protein, in which the nucleotides encoding the hemagglutinin ectodomain are replaced by nucleotides encoding the heterologous protein, said protein containing an epitope of an ectodomain of a protective antigen of an agent infectious virus other than influenza or an antigen that is associated with a disease, said fusion protein is anchored by the C-terminus, so that both the influenza hemagglutinin and the fusion protein are expressed and incorporated into the attenuated chimeric influenza virus.

[0243] The present invention provides an attenuated chimeric influenza virus, comprising a bicistronic enveloped influenza virus NA segment, comprising: (a) a first open reading frame encoding an avian influenza neuraminidase protein, and (b ) a second open reading frame encoding a neuraminidase fusion protein, in which nucleotides encoding the neuraminidase ectodomain are replaced by nucleotides encoding heterologous protein, said protein containing an epitope of an ectodomain of a protective antigen of a different infectious agent of influenza or an antigen that is associated with a disease, said fusion protein is anchored by the N-terminus, so that both the influenza neuraminidase and the fusion protein are expressed and incorporated into the attenuated chimeric influenza virus.

[0244] In some embodiments, the attenuated chimeric influenza virus of paragraphs 221-224 comprises an enveloped NS1 gene segment encoding a modified NS1 protein that reduces the cellular interferon antagonist activity of the virus. In some embodiments, the attenuated chimeric influenza virus of paragraphs 221-224 comprises an HA segment having an open reading frame modified to remove the hemagglutinin polybasic cleavage site. In other embodiments, the attenuated chimeric influenza virus of paragraph 223, in which the first open reading frame is modified to remove the hemagglutinin polybasic cleavage site.

[0245] The present invention provides recombinant DNA molecules encoding the NA segment of paragraphs 221 and 224. The present invention further provides recombinant DNA molecules encoding the HA segment of paragraphs 222-223.

[0246] The present invention provides methods for propagating the attenuated chimeric influenza viruses of paragraphs 221-225, comprising cultivating the attenuated chimeric influenza viruses in an embryonated egg or a cell line that is susceptible to infection by avian influenza viruses. The present invention further provides methods for producing an immunogenic formulation, the method comprising: (a) propagating the attenuated chimeric influenza virus of paragraphs 211 and 2221-225 in an embryonated egg or a cell that is susceptible to infection by attenuated influenza viruses ; and (b) collecting the progeny virus, wherein the virus is grown in sufficient quantities and under sufficient conditions that the virus is free from contamination such that the progeny virus is suitable for use in an immunogenic formulation, e.g. vaccine formulation.

[0247] The present invention provides a chimeric NDV, comprising a packaged genome comprising a nucleotide sequence encoding an F protein - fusion protein having the transmembrane and cytoplasmic domains of an F protein and the ectodomain of an antigen from a different infectious agent. of NDV that is anchored by the C-terminus, so that the F protein - fusion protein is expressed and incorporated into the chimeric NDV.

[0248] The present invention provides a chimeric NDV, comprising a packaged genome comprising a nucleotide sequence encoding an HN fusion protein having the transmembrane and cytoplasmic domains of an HN protein and the ectodomain of an antigen of an infectious agent other than NDV that is anchored by the N-terminus, so that the HN fusion protein is expressed and incorporated into the chimeric NDV.

[0249] In some embodiments, the chimeric NDV genome of paragraphs 213 and 228-229 comprises a nucleotide sequence encoding an F protein, such that the F protein is expressed and incorporated into the chimeric NDV in addition to the F protein - fusion protein. In other embodiments, the nucleotide sequence encoding the F protein - NDV fusion protein replaces the nucleotide sequence encoding the NDV F protein, and the F protein - fusion protein supplies the function of the F protein for the chimeric NDV of paragraph 228.

[0250] In some embodiments, the chimeric NDV genome of paragraphs 212 and 223-224 comprises a nucleotide sequence encoding an HN protein, such that the HN protein is expressed and incorporated into the chimeric NDV. In other embodiments, the nucleotide sequence encoding the HN fusion protein replaces the nucleotide sequence encoding the HN protein of NDV and the HN fusion protein supplies the function of the HN protein for the chimeric NDV of paragraph 229.

[0251] The present invention provides methods for propagating the chimeric NDVs of paragraphs 212 and 228-229, comprising culturing the chimeric NDVs in an embryonated egg or a cell line that is susceptible to NDV infection. The present invention further provides a method for producing an immunogenic formulation, the method comprising: (a) propagating the chimeric NDV of paragraphs 212 and 228-229 in an embryonated egg or cell; and (b) collecting the progeny virus, wherein the virus is grown in sufficient quantities and under sufficient conditions that the virus is free from contamination such that the progeny virus is suitable for use in an immunogenic formulation, e.g. , vaccine formulation.

[0252] The present invention provides embryonated eggs comprising the chimeric viruses of paragraphs 209-210, 212-218 and 228-229. The present invention further provides cell lines comprising the chimeric viruses of paragraphs 209-210,

[0253] The present invention provides an embryonated egg comprising the attenuated chimeric viruses of paragraphs 211 and 221-225. The present invention further provides cell lines comprising the attenuated chimeric viruses of paragraphs 211 and 221-225. The present invention further provides immunogenic formulations comprising the attenuated chimeric viruses of paragraphs 211 and 221-225.

[0254] The present invention provides methods of inducing an immune response to two infectious agents in a bird, the method comprising administering an effective amount of a chimeric avian influenza virus of paragraphs 209-210 and 213-218. The present invention further provides methods of inducing an immune response to two infectious agents in a bird, the method comprising administering an effective amount of a chimeric NDV of paragraphs 212 and 228-229. The present invention further provides methods for inducing an immune response to two infectious agents in an individual, the method comprising administering an effective amount of an attenuated chimeric influenza virus of paragraphs 211 and 221-225. In some embodiments, the subject is a human subject. In other embodiments, the subject is a non-human mammal (e.g., a pig, horse, dog, cat, or bovine). In still other embodiments, the subject is an avian subject.

6. EXAMPLES 6.1 Engineering chimeric avian influenza viruses featuring a Newcastle disease virus epitope

[0255] The following example describes the production of an exemplary chimeric avian influenza virus. In particular, the example describes the engineering of an avian influenza virus, Influenza A/Vietnam/I 203/04 (H5N1), to express and incorporate into its virion a fusion protein comprising the transmembrane and cytoplasmic domains of the NA protein of avian influenza virus and ectodomain of the HN protein of NDV. The fusion protein functionally replaces the NA protein of avian influenza viruses.

6.1.1 Materials and Methods 6.1.1.1 Construction of plasmids

[0256] All plasmid constructions for use in plasmid-only rescue of recombinant viruses were cloned using the same strategy. Full-length cDNAs from viral segments were amplified using PCR with primers that included SapI restriction sites, which allowed an insertion of the PCR product into the SapI sites of the pPo1I-SapI-Rb plasmid (Flandorfer et al, 2003, J. Virol. 77:9116-9123; Nakaya et al, 2001, J. Virol. 75:11868-11873; both of which are incorporated herein by reference in their entirety). Sequences of all PCR inserts were confirmed (Mount Sinai DNA Sequencing Facility, NY), and nucleotide changes that were introduced by PCR were corrected using a QuickChange XL Site-Directed Mutagenesis Kit (Stragene, La Jolla, CA) when appropriate. The GenBank sequences of Influenza A/Vietnam/1203/04 (H5N1), Influenza A/WSN/33 (WSN), and NDV are provided in Table 2 Table 2. GenBank accession numbers of virus segments

6.1.1.2 Construction of chimeric viral segment

[0257] A cDNA encoding the HN ectodomain of B1 of NDV and the cytoplasmic tail (CT) and transmembrane (TM) domains of the neuraminidase (NA) of influenza A/WSN/33 (A/Vietnam/I 203/04 - A/WSN/33 NA(CT+TM)-HN DE B1 of NDV (ecto)) was constructed using recombinant techniques well known in the art. The construct encodes 19 nucleotides of the 3' non-coding region of the WSN NA vRNA, nucleotides encoding amino acids 1-36 (108 nucleotides) of a NA coding region, corresponding to the cytoplasmic and transmembrane tail domains of the NA protein plus the first amino acid of the NA ectodomain, followed by nucleotides encoding amino acids 51-568 of the NDV B1 HN protein (HN ectodomain), two sequential stop codons, untranslated nucleotides 157 of the WSN NA reading frame, and a non-translated region. 5' encoding of the WSN vRNA (FIG.1).

6.1.1.3 Construction of plasmid constructs encoding chimeric H5N1 NDV

[0258] Plasmid constructs were created in order to produce, by plasmid-only rescue, a chimeric virus based on H5N1 (the host virus) engineered to present a surface NDV glycoprotein. The segment of H5N1 encoding the NA surface glycoprotein was selected to be replaced with a recombinant segment comprising a nucleotide sequence encoding the CT and TM domains of the NA protein plus the first amino acid of the NA ectodomain of A/WSN/33 and the ectodomain of the NDV B1 HN protein. The fusion protein, A/Vietnam/I 203/04 -A/WSN/33 NA(CT+TM)-HN of NDV B1 (Ecto), provides neuraminidase activity for the chimeric avian influenza virus. See FIG. 1 for a schematic of the chimeric segment.

[0259] The remaining seven segments of H5N1 listed in Table 2 (NS, M, NP, HA, PA, PB1 and PB2) were cloned into pPol1 to produce pPol1VN1203-NS, pPol1 VN1203-M, pPol1VN1203-NP, pPol1VN12Q3-HA , pPol1VN1203-PA, pPol IVN 1203 - PB1 and pPol1VN1203-PB2, respectively. To ensure attenuation of the H5N1 chimeric virus, the H5N1 HA-coding segment was altered to convert the native polybasic amino acid sequence immediately preceding the HA cleavage site (nucleotides 1013-1039 of the H5N1 HA-coding sequence) to a sequence consensus based on avirulent avian strains of influenza A H5. The amino acid sequence in this region was changed from QRERRRKKRG (SEQ ID NO: 11; amino acids 2-11 of SEQ ID NO: 14) to QRETRG (SEQ ID NO: 12; amino acids 2-7 of SEQ ID NO: 16), replacing the amino acids underlined with threonine (FIG.2). The codon usage in this region was further altered to reduce the number of adenosine residues in order to minimize the possibility of reintroduction of adenosine residues into this sequence by polymerase slippage and the resulting introduction of basic amino acid residues into the HA cleavage site. . Only synonymous mutations were introduced into the avirulent HA sequence (FIG. 3). The resulting segment encoding the altered HA glycoprotein, corresponding to low virulence avian influenza A strains, was cloned into a pPol1 plasmid as previously described, pPol1VN1203-HALO. With the exception of PB1 and PB2, the gene products encoded by the H5N1 segments were unchanged from the GenBank sequences. The sequences of PB1 and PB2 were altered as a result of the introduction of the SapI restriction sites. A nonsynonymous substitution with the nucleotide guanine at position 32 of the PB1 coding sequence resulted in a lysine to arginine mutation; substitution with the nucleotide thymine at position 1393 of the PB2 coding sequence resulted in a proline to serine mutation. All H5N1 HA gene products have an adenosine residue at position 4 of the vRNA.

[0260] In addition to the plasmid construct encoding wild-type H5N1 NS, pPol1VN1203-NS, three pPol1 constructs differently encoding truncated versions of the H5N1 NS gene segment were also generated. Additional constructs encoding altered versions of the NS segment may be of use in further attenuation of the resulting chimeric virus (see, for example, U.S. Patent 6,669,943, which is incorporated herein by reference in its entirety). The three constructs varied in the number of amino acids of the NS1 protein (from the amino terminus) that are expressed by the plasmid construct. pPol1VN1203 NS1-126, pPol1VN1203 NS1-99 and pPol1VN1203 NS1-73 thus encode only the first 126, only the first 99 and only the first 73 amino acids as counted from the amino terminus of the wild-type NS1 protein, respectively. Mutagenesis to generate truncated constructs does not affect the NEP open reading frame (FIG. 4).

6.1.1.4 Rescue of infectious viruses from plasmid constructs

[0261] Recombinant chimeric viruses of the invention are rescued by any means described herein or known in the art. For example, 293T, HEp-2 or A549 cells can be transfected with eight of the described pPol1 plasmids, selected to effect a desired level of viral attenuation and so that all eight segments are represented, i.e., the cells are transfected with pPol1 VN WSN-NA(CT+TM)-HN of B1 of NDV(ecto); pPol1VN1203-HA or pPol1 VN1203-HALO; pPol1VN1203-NS, pPol1VN1203 NS1-126, pPol1VN1203 NS1-99 or pPol1VN1203 NS1-73; pPol1VN1203-M; pPol1VN1203-NP; pPol1VN1203-PA; pPol1 VN1203-PB1 and pPol1 VN1203-PB2. The cells are further transfected with eukaryotic expression plasmids encoding NA, PA, PB1 and PB2, which are required for replication and transcription of the vRNAs. After overnight incubation, the transfected cells can be co-cultured with chicken embryo fibroblasts to amplify the virus produced. After another 2- to 3-day incubation, the co-culture supernatant can be injected into the allantoic cavities of 9- or 10-day-old embryonated chicken eggs for propagation. For attenuated viruses, 7-day-old eggs that do not have a competent interferon system can be used. Virus growth can be confirmed by testing the collected allantoic fluid for hemagglutination according to standard protocols known in the art.

6.2 Engineering chimeric Newcastle disease virus presenting a foreign epitope

[0262] The following example describes the production of exemplary chimeric NDVs. In particular, the example describes the engineering of a chimeric NDV to express and incorporate into its virion a fusion protein comprising the transmembrane and cytoplasmic domains of a required NDV protein and the ectodomain of an avian influenza virus. The example demonstrates that such a chimeric virus induces protection against subsequent infection by both influenza viruses and NDV.

[0263] The example also describes the engineering of an exemplary NDV to express and incorporate into its virion a fusion protein comprising the cytoplasmic domain of the NDV F protein and the ectodomain and transmembrane domain of human keratinocyte growth factor receptor ( KGFR).

6.2.1 Materials and Methods 6.2.1.1 Cell lines

[0264] MDCK, HEp-2 and A549 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin. The full-length cDNA of the B1 Hitchner strain of NDV has been published under GenBank accession number AF375823 (Nakaya et al., 2001, J. Virol. 75:11868-11873, which is incorporated herein by reference in its entirety).

6.2.1.2 Construction of Plasmids

[0265] The engineering of recombinant NDV cDNA to encode a foreign protein has been described (Nakaya et al., 2001, J. Virol. 75:11868-11873). Briefly, the full-length NDV cDNA is introduced into a plasmid between the T7 promoter and the hepatitis delta virus (HDV) ribozyme and T7 terminator to create pNDV/B1. The NDV cDNA has an engineered XbaII site between the P and M genes that allows the introduction of foreign sequences as an extratransciptional unit into the NDV genome (FIG. 5). All inserted genes are engineered to sequentially contain a gene ending; 5'-TTAGAA AAAA-3' (SEQ ID NO: 18); intercistronic T nucleotide; and the initial gene sequence; 5'-ACGGGTAGAA-3' (SEQ ID NO: 19) (the GE/GS sequence).

[0266] rNDV/B1 -KGFR, rNDV/B1 -KGFR/F-CT, and rNDV/B1 -H7HA/F- TMCT viruses were generated by reverse genetics from copies of full-length cDNA derived from the B1 Hitchner strain of NDV . To construct these viruses, the KGFR or H7HA (HA protein of influenza A subtype H7N2) ORF was cloned as an extratranscriptional unit between the P and M genes of NDV/B1 cDNA, as described for other ORFs (Nakaya et al, 2001 , J. Virol. 75:11868-11873 and Nakaya et al, 2004, J. Virol. 78:9366-9375, both of which are incorporated herein by reference in their entirety). KGFR and H7HA are both transmembrane proteins, each comprising a TM and CT domain. In the KGFR/F-CT construct, the CT domain of the KGFR protein was replaced by that of the NDV F protein. In the H7HA/F-TMCT construct, the TM and CT domains of the H7HA protein were replaced with those of the NDV F protein. Recombinant NDV viruses were rescued from cDNA and propagated using well-known standard techniques (see, for example, Swayne et al, 2003, Avian Dis. 47:1047-1053 and Nakaya et al, 2001, both of which are incorporated herein by reference in its entirety). The insertion of new transcriptional units into the recombinant viruses was confirmed by reverse transcription PCD followed by sequencing analysis.

[0267] For example, the ectodomain (ECTO) of the H5 HA gene was produced by PCR using the following primers (which include the GE/GS sequence): Nhel- H5HA P, 5'- CG GCT AGC TTAGAAAAAA T ACGGTAGAA GTGAA ACTAGT CC GCC ACC ATG GAA AGA ATA GTG ATT GCC TTT GCA-3' (SEQ ID NO:20) and HpaI- H5HA P, 5'-CG GTT AAC CTG ATA AGC CCC CAT TGA TTC TAA T-3' (SEQ ID NO:21). The H5 HAecto PCR fragment was digested with Nhel and Hpal and cloned into pSL1180 (Amersham Pharmacia Biotech) (pSLH5HAecto). The TM and CT of the NDV F gene were also amplified by PCR using the following primers, HpaI- NDVF(TM+CYTO) P, 5'-CG GTT AAC CTC ATT ACC TAT ATC GTT TTG ACT-3' (SEQ ID NO:22), Sacl-Nhel-NDVF(TM+CYTO) M, 5'-CG GAG CTC AA GCT AGC TTA TCA CAT TTT TGT AGT GGC TCT CAT CTG-3' (SEQ ID NO:23). To fuse with H5 HAecto, the TM and CT of the NDV F gene were digested with Hpal and SacI and then cloned into pSLH5HAecto to obtain the hybrid fusion gene. Finally, the plasmid containing the hybrid HA H5 gene was digested with Nhel and cloned between the P and M genes of the rNDV cDNA.

6.2.1.3 Western Blot and Biological Analysis

[0268] Viruses from cell or allantoic extracts were purified by ultracentrifugation through a 30% sucrose cushion. Levels of incorporated protein were monitored by Western blot analysis using specific antibody and routine techniques.

[0269] The ability of chimeric NDV to present the non-viral protein KGRF in vivo was determined by immunizing BALB/c mice with 3 X107 pfu of the chimeric virus intraperitoneally, followed by a booster immunization using

[0270] An in vivo system was designed to evaluate whether immunization with rNDV comprising the hybrid H7HA/F-TMCT was able to provide protection against subsequent infection by H7 or NDV. Two-week-old chicks were immunized by eye drop method with 100 µl of three vaccines, rNDV, rNDV - H7HA/F-TMCT and placebo. At 4 weeks of age, 100 µl comprising a mean infectious dose of 105.1 of the HP AIV embryo (A/Steele/ACC- 10/59 [H7N7]) was administered through the choanal cleft. Birds were observed for signs and lesions of HP AIV infection. Mortality was recorded, and all survivors were euthanized by sodium pentobarbital (100 mg/kg) at 6 weeks of age.

6.2.2 Results 6.2.2.1 Presentation of KGFR by chimeric NDV expressing KGFR or KGFR/F- CT

[0271] Chimeric viruses rNDV/B1 -KGFR and rNDV/B1 -KGFR/F-CT were cultured in the allantoic cavity of 10-day-old embryonated chicken eggs. Purified viruses were tested for the presence of KGFD or KGFR/F-CT by Western blot analysis using a murine anti-KGFR antibody. A positive response was detected in samples isolated from eggs inoculated with rNDV/B1 -KGFR/F-CT but not with rNDV/B1-KGFR (FIG. 6).

[0272] Each of these chimeric viruses was also used to immunize three BALB/c mice. Sera from immunized animals were tested for the presence of KGFR antibodies. Animals immunized with rNDV/B1-KGFR virus did not develop detectable levels of KGFR antibodies using this test. In contrast, all three animals immunized with rNDV/B1-KGFR/F-CT virus were positive by this test for the presence of KGFR antibodies.

6.2.2.2 Protection against H7 infection by immunization with rNDV-H7HA/F-TMCT

[0273] The TM and CT domains of wild-type H7HA were replaced with the TM and CT domains of the NDV F protein to generate a hybrid HA protein, H7HAecto-NDV/F(TM+ CT). In a Western blot analysis, both control rNDV expressing the complete H7HA ORF, rNDV-H7HA, and chimeric rNDV expressing H7HAecto- NDVZF(TM + CT) hybrid, rNDV- H7HAecto-NDV/F(TM + CT), generated a positive reaction for the H7 antibody; however, the rNDV-H7HAect0-NDV/F(TM + CT) signal was visible many times stronger (FIG. 7). When chicks immunized once with rNDV-H7HAect0-NDV/F(TM + CT) were subsequently challenged with a lethal dose of H7 influenza, 9 out of 10 (90%) of the immunized chicks survived. When chicks immunized once with rNDV- H7HAecto- NDV/F(TM+ CT) were subsequently challenged with a lethal dose of NDV, 10 out of 10 (100%) immunized chicks survived.

6.3 Engineering Chimeric Newcastle Disease Virus Presenting a Foreign Epitope

[0274] The following example describes the production of modified chimeric NDVs. In particular, a recombinant NDV was produced to improve the virulence of the NDV backbone used in Example 6.2. The example shows that the improved virulence of rNDV also improves the immunogenicity of immunogenic formulations comprising chimeric viruses based on rNDV.

6.3.1 Materials and Methods

[0275] Unless otherwise specified, all materials and methods described in the section are identical to those described and exemplified in Example 6.2, above. 6.3.1.1 Generation of rNDVs with a modified cleavage site in their F proteins

[0276] Recombinant NDV virus rNDV/F2aa and rNDV/F3aa viruses, which have two or three amino acid mutations in the F cleavage site of NDV Hitchner strain B1 were generated by reverse genetics. Briefly, to generate rNDV/F2aa, the PCR fragment was generated using primers, forward: F2aa-1(+) 5'-GGA TCC CGG TTG GCG CCC TCC AGG (SEQ ID NO:24), and reverse F2aa-1( -) 5'-AAG GCG CCt CTG TCT CCg CCC TCC AGA TGT AGT CAC AG-3' (SEQ ID NO:25) and the full-length NDV B1 clone, plasmid pT7NDV/B1, as a template. The following PCR fragment was generated using primers, forward: F2aa-2(+) 5'-GGc GGA GAC AGa GGC GCC TTA TAG GCG CCA TTA TTG G-3' (SEQ ID NO:26), and reverse F2aa-2 (-) 5'-CCA TAT TCC CAC CAG CTA GAT TGT-3' and (SEQ ID NO:27) and pT7NDV/B1 as a template. The nucleotides shown in lowercase are changed to modify the amino acid sequence of the NDV/B1 strain F protein cleavage site (GGRQGR^X) to GRRQRR^L. These two overlapping PCR fragments (the overlap is underlined in the primer sequences) were combined by PCR using primers, F2aa-1(+) and F2aa-2(-). The resulting PCR fragment, which contains the complete F gene, was cloned into pSL1180 (Amersham Pharmacia Biotech) and designated pSLF2aa. The Stul-Notl fragment (nt 4646 to 4952) from pSLF2aa was extracted to replace the corresponding fragment in the pT7NDV/B1 plasmid, resulting in the formation of the pT7NDV/F2aa plasmid, which was used to generate the rNDV/F2aa virus by reverse genetics. For generation of rNDV/F3aa, PCR mutagenesis was performed by the same strategy as described above using primers, forward, F3aa-1(+) 5'-GGA TCC CGG TTG GCG CCC TCC AGG-3' (SEQ ID NO:28) ; reverse, F3aa-1(-) 5'-AAa GCG CCt CTG TCT CCg CCC TCC AGA TGT AGT CAC AG-3' (SEQ ID NO:29); front, F3aa-2(+) 5'-GGc GGA GAC AGa GGC GCt TTA TAG GCG CCA TTA TTG G-3' (SEQ ID NO:3Q); reverse F3aa-2(-) 5'-CCA TAT TCC CAC CAg CTA GAT TGT-3' (SEQ ID NO:31) (transformed nucleotides are indicated with lowercase letters and pT7NDV/B1 as a template. These two overlapping PCR fragments (the overlapping region is underlined in the primer sequences) were combined by PCR using primers F3aa-1(+) and F3aa-2(-), resulting in modification of the cleavage site from GGRQGR^L to GRRQRR^F. of Stul-Notl (nt 4646 to 4952) from pSLF3aa was extracted to replace the corresponding fragment in the pT7NDV/B1 plasmid resulting in the formation of the pT7NDV/F3aa plasmid, which was used to generate the rNDV/F3aa virus.

6.3.1.2 Generation of a fusogenic rNDV vector expressing the chimeric H7HA protein

[0277] To construct the chimeric H7HA gene as an extratranscriptional unit of the rNDV/F3aa genome, the fragment containing the transmembrane (TM) and cytoplasmic tail (CYTO) of the NDV F gene was initially produced by PCR using primers, HpaNDV F(TM+CYTO)P, 5'-cgGT TAA CCT CAT TAC CTA TAT CGT TTT GAC T-3' (SEQ ID NO:32) and SacNheNDVF(TMH-CYTO)M, 5'-cg GAG CTC AAG CTA GCT TAT CAC ATT TTT GTA GTG GCT CTC ATC TG-3' (SEQ ID NO:33) and the plasmid containing the NDV F gene as a template. This PCR product was digested with Sac I and Hpa I and then cloned into the plasmid, pNhe-NDV-GE/GS possessing the gene termination and gene starting signal of NDV, resulting in the formation of plasmid, pNhe-NDV- GE/GS- NDVF(TM+CYTO). As the next step, allowing the connection of the fragment containing the H7HA ectodomain with the TM and CYTO region fragment of NDV F, the H7HA ectodomain was produced by PCR using the primers, SpeH7(ECTO)P, 5'-cgACT AGT CCG CCA CCA TGA ACA CTC AAA TTC TGG CAT TCA T-5' (SEQ ID NO:34), HpaH7(ECT0)M, 5'-cgG TTA ACG TCT TTG TAT CCA CTA CTC AAT TTC AC-3' (SEQ ID NO:35) and plasmid containing H7HA gene from A/galinha/NY/13142-5/94(H7N2) as a template. This PCR product was digested with Spe I and Hpa I and then inserted into the cassette plasmid, pNhe-NDV-GE/GS-NDVF(TM+CYTO). In a final step, the cassette plasmid, pNhe-NDV- GE/GS- NDV F(TM+CYTO) was digested with Nhe / to cut out the chimeric H7HA gene. This fragment DNA was cloned between the P and M genes of pT7NDV/F3aa, forming chimeric pT7NDV/F3aa-H7. The rNDV/F3aa virus expressing the chimeric H7HA protein was then rescued from the chimeric pT7NDV/F3aa-H7 using methods described, supra.

6.3.1.3 Viral growth kinetics

[0278] rNDV/B1, rNDV/F2aa, rNDV/F3aa, rNDV/B1 -H7, or rNDV/F3aa- H7 chimeric viruses (100 PFU/egg) were inoculated into the allantoic fluids of 10-day-old embryonated chicken eggs to determine viral titers at different time points (24 hours, 48 hours, and 72 hours). The 5% tissue culture infective dose (TCID50) of each virus present in the allantoic fluid was determined by immunofluorescence testing (IFA). For this purpose, ninety-six-well plates containing Vero cells were infected with serial dilutions of 10

[0279] MDCK cells infected with transfecting influenza virus were fixed and permeabilized with ice-cold methanol. Viral antigens were detected with anti-NDV HN monoclonal antibody (7B1), anti-influenza H1HA monoclonal antibody (2G9), and anti-influenza H5HA polyclonal serum. For analysis of NDV growth and viral protein expression, confluent Vero cells were infected with the recombinant viruses, and collected at different time points (24, 48, and 72 hours). Infected cells were fixed with 2.5% formaldehyde containing 0.1% Triton X-100. Fixed cells were treated with anti-rabbit NDV polyclonal antibody or anti-chicken AIV H7 polyclonal serum, washed, and stained with fluorescin isothiocynate (FITC)-conjugated anti-chicken immunoglobulins (DAKO) to AIV H7HA protein or anti-rabbit immunoglobulins. Texas Red (Molecular Probe) conjugates to NDV viral proteins. Viral protein expression was examined by fluorescence microscopy.

6.3.2.2 Average time of death

[0280] To verify the pathogenicity of recombinant viruses in embryonated chicken eggs, mean death time (MDT) was determined. Briefly, five 10-day-old embryonated chicken eggs were infected with 10-fold serial dilutions of virus. Eggs were incubated at 37°C and monitored twice daily for 7 days. The time to kill the embryos was recorded. The highest dilution that killed all embryos was determined to be the minimum lethal dose. The MDT was calculated as the average time for the minimum lethal dose to kill all embryos.

6.3.2.3 Immunization and challenge of chickens

[0281] Leghorn White chickens were vaccinated once or twice by drops in the eye in the conjunctival sac with mean infectious doses 105.7-6.1 chicken embryo (EID5o) of rNDV/F3aa- chimericH7, or twice with 105. 7-6.3 EID50 of parental NDV/B1 (pNDV), or twice with sterile tissue culture medium (placebo) at 2 and 4 weeks of age. At 6 weeks of age, chickens were challenged intranasally with the Fontana strain of velogenic NDV (vvNDV)(105.1 EID50 per HPAI (105.1 EID50 per bird) or A/Human/Steele./59 (H7N7) HPAI ( 105.1 EID50 per bird) Survivors were bled and sacrificed within 14 days of challenge.Hemagglutination inhibition (HI) serological titers were determined using standard procedures.

6.3.3 Results 6.3.3.1 Generation of fusogenic rNDV mutants

[0282] To improve the fusogenic characteristics of the rNDV backbone, two rNDV mutants, rNDV/F2aa and rNDV/F3aa viruses, were developed in which the F protein cleavage site was replaced with one of two multibasic cleavage site variants. , which can be activated by ubiquitously expressed proteases (e.g., furin proteases) (Fig. 8A). Infection of chicken embryo fibroblast (CEF) cells with rNDV/F2aa and rNDV/F3aa, and with rNDV/B1, resulted in the formation of syncytia in the absence of exogenously added protease (Fig. 8B). In addition, rNDV/F3aa induced syncytia more rapidly in CEF cells than rNDV/F2aa. It has therefore been postulated that enhanced expansion of the virus in immunized animals may increase immunogenicity against inserted foreign protein. Therefore, fusogenic rNDV/F3aa was selected as a backbone vector to develop a bivalent vaccine designed to protect poultry against AIV and NDV.

6.3.3.2 Analysis of mean death time of rNDV platform vectors in embryonated chicken eggs.

[0283] NDV can be classified as highly virulent (velogenic), intermediate (mesogenic), or non-virulent (lentogenic) on the basis of its pathogenicity to chickens. Since the presence of an F protein with a multibasic cleavage site is known to be a virulence factor of NDV, we evaluated the pathogenicity of rNDVs with modified F protein in 10-day-old embryonated chicken eggs. The mean death time (MDT) of chicken embryos infected with in vivo virulent NDV correlates. Lentogenic strains (causing asymptomatic infection in birds) are characterized by MDTs of greater than 90 hours, mesogenic strains (causing respiratory disease in birds) have MDTs between 60 to 90 hours, and velogenic strains (causing severe disease in birds) have MDTs below 60 hours. The MDT of rNDV/F2aa was indicative of a viverogenic strain while that of rNDV/F3aa was typical of a mesogenic strain. Neither of these strains had MDTs typical of a highly pathogenic (velogenic) strain (Table 3). Table 3. MDT of rNDVs in embryonated chicken eggs

[0284] Based on the data in this table, rNDV/F3aa vector would not pose a danger to birds and is therefore suitable as a backbone for developing a bivalent vaccine for the protection of poultry against AIV and NDV.

6.3.3.3 Generation of a fusogenic rNDV vector expressing the AIV HA protein ectodomain.

[0285] The gene encoding the H7HA protein from A/galinha/NY/ 13142-5/94(H7N2) was incorporated into the rNDV/F3aa vector as described above, resulting in the formation of chimeric rNDV/F3aa-H7 (Fig. 9A). The kinetic growth of chimeric rNDV/F3aa-H7 in embryonated chicken eggs was compared to that of parenteral rNDV/F3aa (Fig. 9B). Viruses expressing the chimeric H7HA protein grew more slowly than viruses without the insertion and maximum titers were about log lower. Interestingly, the MDT of this virus was that of a ladogenic strain (128 ~ 140 hours) (Table 3). Protein expression of chimeric H7HA from rNDV/F3aa-H7 chimeric was confirmed by western blotting of infected Vero cells 36 hours postinfection (Fig. 9C).

6.3.3.4 Enhanced incorporation of AIV H7HA protein into rNDV virions.

[0286] To determine whether expression of the chimeric H7HA protein containing the heterologous transmembrane regions and cytoplasmic tail of the NDV F protein would be associated with increased incorporation into rNDV virions, rNDV/B1-H7 virions and rNDV/F3aa-H7 virions chimeric were purified as described in § 6.3. The amounts of H7HA protein or NDV viral protein from rNDV/B1-H7 or chimeric rNDV/F3aa-H7 were measured by western blotting using anti-chicken AIV H7 polyclonal antibody or anti-rabbit NDV polyclonal sera. As expected, incorporation of chimeric H7HA protein into rNDV virions was significantly increased when compared with that of wt H7HA protein (Fig. 9D). These data suggest that the transmembrane and cytoplasmic tail regions of the NDV F protein play a major role in enhancing incorporation of the foreign protein onto the viral surface.

6.3.3.5 Immunization and challenge of chickens.

[0287] Following one or two vaccinations with rNDV/F3aa-chimericH7, 50-80% of chickens had hemagglutination inhibition (HI) titers for H7 AIV and 90-100% of chickens had HI titers for NDV (Table 4 A and B). While all chickens immunized twice with parenteral NDV/B1 (pNDV) showed HI titers for NDV but none had titers for H7 AIV. All birds infected with sterile tissue culture medium (placebo) lacked HI titers for both viruses. When challenged with vvNDV, 100% of chickens immunized with rNDV/F3aa- chimeric H7 and pNDV were protected. By comparison, 90% of chickens vaccinated with chimeric rNDV/F3aa-H7 were protected against HPAI H7 virus, but none of the chickens vaccinated with pNDV were protected from HPAI H7 virus. By contrast, 100% and 70% of placebo-infected birds died when challenged with vvNDV and HPAI H7 viruses, respectively. Survivors mounted an amnestic response evident as a fourfold or greater elevation in HI titer to the respective challenge virus except for the three survivors in the placebo-HP AI H7 virus challenge group who had no serological evidence of being infected. Table 4A HI serology of chickens immunized with chimeric viruses before Challenge *n=10 birds per group, 1X=one vaccination, 2X=2 vaccinations

[0288] The publication entitled "Engineered Viral Vaccine Constructs with Dual Specificity: Avian Influenza and Newcastle Disease", by Man-Seong Park et al, in PNAS 103:8203-8208(2006) is incorporated herein by reference in its entirety.

6.4 Equivalences

[0289] The present invention is not limited in scope by the specific embodiments described which are intended to be simple illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, several modifications of the invention, in addition to those shown as evident to those skilled in the art from the preceding description and accompanying drawings. Such modifications are intended to be within the scope of the attached claims.

[0290] Throughout this application several publications were cited. Its contents are hereby incorporated by reference into this application in its entirety for all purposes.

Claims (24)

1. Newcastle disease chimeric virus (NDV), characterized by the fact that it comprises an enveloped genome comprising a nucleotide sequence encoding an F fusion protein, wherein the F fusion protein has an epitope of an ectodomain of a protective antigen of a infectious agent other than NDV or a disease-associated antigen anchored by its C-terminus to the transmembrane and cytoplasmic domains of an NDV F protein, such that the F fusion protein is expressed and incorporated into the chimeric NDV, and in which the antigen is not a paramyxovirus antigen; wherein the chimeric NDV comprises a functional NDV F protein in addition to the F fusion protein, wherein the sequence encoding the F fusion protein is inserted between the P and M genes of the NDV genome, and wherein there is no no deletion or inactivation of native NDV proteins. 2. Chimeric NDV according to claim 1, characterized in that the genome comprises a nucleotide sequence encoding an F fusion protein, wherein the F fusion protein comprises the transmembrane and cytoplasmic domains of an NDV F protein and a ectodomain of a protective antigen from an infectious agent other than NDV that is anchored by the C-terminus of the antigen. 3. Chimeric NDV according to claim 1, characterized in that the genome comprises a nucleotide sequence encoding an F fusion protein, wherein the F fusion protein comprises the transmembrane and cytoplasmic domains of an NDV F protein and a antigen associated with a disease that is anchored by the C-terminus of the antigen. 4. Chimeric NDV according to any one of claims 1, 2 or 3 characterized in that the antigen is a viral antigen, a bacterial antigen, a protective antigen associated with a parasite or a fungal antigen, preferably wherein the viral antigen is an antigen from adenoviridae, herpesviridae, leviviridae, poxviridae, picornaviridae, papovaviridae, reoviridae, preferably wherein the bacterial antigen is an antigen from bacteria of the Aquaspirillum family, Azospirillum family, Azotobacteraceae family, Bacteroidaceae family, Bartonella species, Bdellovibrio family, Campylibacter species, Chlamydia species, clostridium, Enterobacteriaceae family, Gardinella family, Haemophilus influenzae, Halobacteriaceae family, Helicobacter family, Legionallaceae family, Listeria species, Methylococcacea family, Neisseriaceae family, Oceanospririllum family, Pasteurellaceae family, Pneumococcus species, Pseudomonas species, Rhizobiaceae family, Spirillum family, family Spirosomaceae, Staphylococcus, Streptococcus, family Yersinia, or family Vampirovibrio; preferably wherein the protective antigen associated with a parasite is an antigen from an amoeba, a malaria parasite, Plasmodium, Trypanosoma cruzi; and preferably wherein the fungal antigen is an antigen from fungi of Absidia species, Aspergillus species, Ascomycetes, Basidiomycetes, Basidibolus ranarum, Blastomyces dermatitidis, Candida species, Coccidioides immitis, Conidiobolus species, Cryptococcus neoformans, Cunninghamella species, dermatophytes, Deuteromycetes, Histoplasma capsulatum , Microsporum gypseum, Mucor pusillus, Oomycetes, Paracoccidioides brasiliensis, Pseudallescheria boydii, Rhinosporidium seeberi, Pneumocystis carinii, Rhizopus species, Saccharomyces species, Sporothrix schenckii, or Zygomycetes. 5. Chimeric Newcastle disease virus (NDV), characterized by the fact that it comprises an enveloped genome comprising a nucleotide sequence encoding an NDV F fusion protein, wherein the F fusion protein comprises the transmembrane and cytoplasmic domains of an F protein of NDV and an ectodomain of a hemagglutinin antigen of an influenza virus that is anchored by the C-terminus of the antigen, such that the F fusion protein is expressed and incorporated into the chimeric NDV, wherein the influenza virus is an influenza virus poultry house; in which there is no deletion or inactivation of native NDV proteins. 6. Chimeric NDV according to claim 1, characterized in that the antigen is an influenza virus hemagglutinin antigen. 7. Chimeric NDV according to claim 5, characterized by the fact that the influenza virus is H7N2. 8. Chimeric NDV according to claim 5, characterized by the fact that the influenza virus is A/hen/NY/13142. 9. Chimeric NDV according to any one of claims 1 to 8, characterized in that the genome comprises a nucleotide sequence encoding an F protein, such that the F protein is expressed and incorporated into the chimeric NDV. 10. Chimeric NDV according to claim 9, characterized by the fact that the F protein is genetically modified at the cleavage site, such that fusogenic activity is increased, and wherein the genetically modified cleavage site comprises a multibasic cleavage site of the amino acid sequence GRRQRRL (F2aa) and GRRQRRF (F3aa). 11. Chimeric NDV according to any one of claims 1 to 10, characterized in that the transmembrane and cytoplasmic domains of the F fusion protein are from the LaSota strain of NDV. 12. Chimeric NDV according to any one of claims 1 to 11, characterized in that the transmembrane and cytoplasmic domains of the F fusion protein are from the B1 Hitchner strain of NDV. 13. Chimeric NDV according to any one of claims 1, 4 to 12, characterized in that the F fusion protein either contains no amino acid residue from the ectodomain of the F protein, or the F fusion protein contains a 14. Chimeric NDV according to any one of claims 1 to 12, characterized in that the F fusion protein contains no amino acid residues from the ectodomain of the F protein. 15. Chimeric NDV according to claim 13, characterized by the fact that the F fusion protein contains a fragment of the F protein ectodomain that does not retain the activity of the F protein ectodomain. 16. Chimeric NDV according to claim 15, characterized by the fact that the F protein ectodomain fragment is between 1 and 15 residues of the ectodomain of an F protein and are immediately adjacent to the transmembrane domain of the F protein. 17. Chimeric NDV according to any one of claims 1 to 16, characterized in that the chimeric NDV has a LaSota NDV strain dorsal structure. 18. Chimeric NDV according to any one of claims 1 to 16, characterized by the fact that chimeric NDV has a dorsal structure B1 Hitchner strain of NDV. 19. Chimeric NDV according to any one of claims 1 to 18, characterized in that the sequence encoding the F fusion protein is inserted between the P and M genes of the NDV genome. 20. Chimeric NDV according to any one of claims 1 to 19, characterized in that the chimeric NDV is attenuated. 21. Immunogenic composition characterized by the fact that it comprises the chimeric NDV, defined in any one of claims 1 to 20. 22. Method for producing an immunogenic formulation, characterized by the fact that it comprises: (a) propagating the chimeric NDV defined in any one of claims 1 to 20 in an embryonated egg or a cell line that is susceptible to an NDV infection; It is 23. Use of the chimeric virus defined in any one of claims 1 to 20, characterized by the fact that it is in the preparation of a medicament for use in inducing an immune response to influenza viruses and NDV in a bird. 24. Use of the chimeric virus defined in any one of claims 1 to 20, characterized by the fact that it is in the preparation of a medicament for use in inducing an immune response to influenza viruses and NDV in a human.