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  Vectors based on recombinant defective viral genomes and their use in the formulation of vaccinesUSPTO Application #: 20070048862Title: Vectors based on recombinant defective viral genomes and their use in the formulation of vaccines Abstract: The vectors comprise a recombinant defective viral genome expressing at least one antigen suitable for the induction of systemic and secretory immune responses or an antibody conferring protection against an infectious agent. The defective viral genome comprises the genome of a parental virus having the viral replicase recognition signals located on ends 3′ and 5′, further comprising internal deletions, and wherein said defective viral genome depends on a helper virus for its replication and encapsidation. These vectors are suitable for the forming of a recombinant system comprising the aforesaid expression vector, and a helper virus. The system is suitable for the manufacture of mono- and polyvalent vaccines against infectious agents of different animal species, especially pigs, dogs and cats, and as expression vehicles for antibodies protective against infectious agents. (end of abstract) Agent: Wyeth/finnegan Henderson, LLP - Washington, DC, US Inventors: Luis Enjuanes Sanchez, Juan Plana Duran, Sara Alonso Villanueva, M'Luisa Ballesteros Jarreno, Joaquin Castilla Castrillon, Jose Manuel Gonzalez Martinez, Ander Izeta Parmesan, Ana Mendez Zunzunegui, Maria Muntion Saenz, Zoltan Penzes, Jose Manuel Sanchez Morgado, Carlos Miguel Sanchez Sanchez, Cristina Smerdou Picazo, Isabel Sola Gurpegui USPTO Applicaton #: 20070048862 - Class: 435320100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Vector, Per Se (e.g., Plasmid, Hybrid Plasmid, Cosmid, Viral Vector, Bacteriophage Vector, Etc.) Bacteriophage Vector, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20070048862. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This is a division of application Ser. No. 09/155,003, filed Sep. 14, 1998, which is a national stage filing under 35 U.S.C. .sctn.371 of International Application PCT/ES97/00059, filed on Mar. 12, 1997, which claims priority to Spanish Application No. 9600620, filed on Mar. 14, 1996, each of which is incorporated herein by reference. [0002] The vectors comprise a recombinant defective viral genome expressing at least one antigen suitable for the induction of systemic and secretory immune responses. The defective viral genome comprises the parental virus genome having viral replicase recognition signals located on ends 3' and 5' further comprising internal deletions, and wherein said defective viral genome depends on a helper virus for its replication. These vectors are suitable for the forming of a recombinant system comprising (a) the aforesaid expression vector, and (b) a helper virus which supplies functional and structural proteins for the replication and encapsidation of the defective genome. This system is suitable for the manufacture of mono- and polyvalent vaccines against infectious agents of various animal species, especially pigs, dogs and cats. FIELD OF THE INVENTION [0003] This invention relates to a number of vectors based on recombinant defective virus genomes expressing antigens suitable for the induction of systemic and secretory immune responses for the prevention of infections in the mucosae, and to their use with vaccinal purposes together with a suitable helper virus. BACKGROUND OF THE INVENTION [0004] The attainment of recombinant proteins using expression vectors is a well-known fact. In general, prokaryotic and yeast expression systems are highly. efficacious and easy to use, whereas the expression systems used containing superior eukaryotic cells present a number of drawbacks relative to low protein production levels and limitations in the host range. Of the existing expression systems for superior eukaryotic cells, baculovirus-based vectors are the most efficacious in terms of protein production. However, they can only be used in insect cells that, as is known, glycosilate proteins differently from the way animal cells do. In addition, the construction of the recombinant virus takes place through a homologous recombination, which is a laborious technique, especially when numerous genetic variants have to be analyzed. [0005] On the other hand, vectors based on DNA viruses suitable for heterologous gene expression are known. However, the use of DNA-based vectors presents numerous drawbacks for, as they replicate in the nucleus of the host cell and can become integrated in the genome, they are therefore not reliable. On the contrary, the use of RNA-based vectors overcomes the drawbacks associated with the use of DNA viruses because, since they replicate not in the genome of the host cell but in the cytoplasm, replication takes place via RNA and not via DNA, and the possibilities of integration in the genome are very low, making the vectors based on these RNA viruses more reliable. [0006] Also well-known are defective interfering particles (DI) containing the virion capsid and a defective genome, which are deletion subgenomic mutants mostly generated form infectious viral genomes by a replication error. In general, the term "DI particle" refers to defective viruses lacking a region of the RNA or DNA genome, containing the proteins and antigens of the virus, requiring co-infection of the infectious parental virus (helper virus) for replication and which specifically interfere with the homologous helper virus, as they replicate at its expense [Huang and Baltimore, Nature, 226, 325-327 (1970)]. DI genomes arise from genome reorganizations as a result of shifts of the RNA polymerase from one RNA template to another or from one segment of an RNA template to another segment of the same molecule. These DI genomes, once they have been generated, self amplify at the expense of the parental genome or the amplifying virus coding for the proteins involved in replication and encapsidation and which has to compete with defective genomes for such products. [0007] DI particles have been obtained and characterized from some coronaviruses, such as the murine hepatitis virus (MHV), infectious bronchitis virus (IBV), and bovine coronavirus (BCV), although DI particles derived from porcine transmissible gastroenteritis virus (TGEV) have not been described. One of the MHV natural DI particles has been used as the basis for the development of an expression vector in which the exogenous gene is inserted under the control of an internal promoter transcription sequence [Lin and Lai, J. Virol., 6110-6118, October (1993)]. [0008] Generally, known heterologous gene expression vectors based on DI particles have some drawbacks related with their species and target organ specificity and their limited capacity for cloning, that limit their possibilities of use, both in basic research and in research applied to the development of such vectors for therapeutical purposes, including vaccines. [0009] Consequently, there is still need of heterologous gene expression vectors that may successfully overcome the mentioned drawbacks. Specifically, it would be highly advantageous to have available some heterologous gene expression vectors with a high level of safety and cloning capacity and which could be designed so that their species specificity and tropism might be easily controlled. [0010] The present invention provides a solution to the existing problem, comprising a vector based on a recombinant defective viral genome expressing antigens suitable for the induction of an immune response and for the prevention of infections caused by different infectious agents in various animal species. The heterologous gene expression vectors (or DNA sequences) provided by this invention have a high level of safety, as well as high cloning capacity, and may be designed so that their species specificity and tropism may easily be controlled, making such vectors suitable for the formulation of vaccines capable of conferring protection against infections caused by the different infectious agents in various animal species. [0011] Therefore, an object of the present invention is a vector based on a recombinant defective viral genome expressing at least one antigen suitable for the induction of immune response--specifically, a systemic and secretory immune response against infectious agents in various animal species--, or an antibody providing protection against an infectious agent provided with a high level of safety and cloning capacity, and which may be designed so that its species specificity and tropism may be easily controlled. [0012] The defective viral genome which serves as a basis for the construction of the said vector is also an additional objective of this invention. [0013] Another additional object of this invention is a recombinant expression system of heterologous proteins comprising (a) the vector described above and (b) a helper virus that will provide the proteins involved in the replication and encapsidation of the recombinant defective viral genome. [0014] Another additional objective of this invention is vaccines capable of inducing protection against infections caused by different infectious agents in various animal species, comprising the recombinant system described above, together with a pharmaceutically acceptable excipient. These vectors can be uni-, or multivalent, depending on whether the expression vectors present in the recombinant system express one, or more antigens capable of inducing an immune response against one, or more infectious agents, or one or more antibodies providing protection against one or more infectious agents. [0015] Other objects provided by this invention comprise a method for the immunization of animals, consisting of the administration of the said recombinant system or vaccine, as well as a method for the protection of newborn animals from infectious agents that infect the mentioned species. BRIEF DESCRIPTION OF THE FIGURES [0016] FIG. 1 shows the structure of TGEV. The virion is a spherical particle consisting of a lipidic envelope in whose interior is a single-chain, positive-polarity RNA molecule of 28.5 kilobases (kb). This RNA is associated to an N protein forming the nucleocapsid. M and sM structural proteins are included in the membrane. Protein S groups itself into trimers, and is anchored on the external part of the envelope forming the peplomers. [0017] FIG. 2 shows the genomic organization of the four sequenced coronaviruses: MHV, IBV, HCV229E (human coronavirus 229E) and TGEV. The open reading frames coding for each protein are shown to scale. In each genome, the beginning of the RNAs expressing each virus is indicated with an arrow. The number of messenger RNAs (mRNA) expressed by viruses MHV or TGEV may vary depending on the viral strain. In this outline, the TGEV arrows correspond to the mRNAs expressed by strain THER-1. The mRNAs are 3'-coterminal and are numbered in decreasing size order. [0018] FIG. 3 shows the expression of the TGEV genome, strain THER-1. The disposition of the open reading frames (ORF) in the genome is indicated: Pol, polymerase; S, sM, M and N, structural proteins; nsp 3a, 3b and 7, non-structural proteins (protein 3b is not produced with this virus). The genome is transcribed in an RNA of equal length but of negative polarity (-) that will serve as a template for the synthesis of the 7 mRNAs (1 to 7). In each mRNA are represented the common sequence, leader, of the 5' end (square), the polyadenine section at the 3' end and the zone which is translated in each one of them (thick lines). [0019] FIG. 4 shows the evolution of the titer of TGEV THER-1 (A) and PUR46-mar 1CC12 (B) isolates with the passage number at high multiplicity of infection (m.o.i.) in ST cells. [0020] FIG. 5 shows the results of the electrophoretic analysis of the RNAs produced in ST cells infected with THER-1 virus passaged 46 times at high m.o.i. The passage number is indicated above each lane and the bars to the left indicate the position of the molecular weight markers (genomic RNA of the TGEV and GibcoBRL markers), expressed in kb. The bars to the right indicate the TGEV mRNAs and the defective interfering (DI) RNAs. NI, not infected. [0021] FIG. 6 shows the results of the Northern-blot assay of the RNA of ST cells infected with the THER-1 p35 virus. Continue reading... Full patent description for Vectors based on recombinant defective viral genomes and their use in the formulation of vaccines Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Vectors based on recombinant defective viral genomes and their use in the formulation of vaccines patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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