MXPA06011714A - Synergistic attenutation of vesicular stomatitis virus, vectors thereof and immunogenic compositions thereof - Google Patents

Abstract

The present invention broadly relates to the synergistic attenuation of vesicular stornatitis virus (VSV). More particularly, the invention relates to the identification of combined mutation classes which synergistically attenuate the pathogenicitnof VSV vectors in mammals and imrPWnogenic compositions thereof.

Description

SYNERGISTIC ATENUATION OF VESUS VIRUS VIRUS, VECTORS OF THEM AND IMMUNOGENIC COMPOSITIONS OF THEM FIELD OF THE INVENTION The present invention relates generally to the fields of virology, microbiology, infectious diseases and immunology.

More particularly, the invention relates to the synergistic attenuation of vesicular stomatitis virus and vectors thereof, by combining different kinds of mutation.

BACKGROUND OF THE INVENTION The vesicular stomatitis virus (VSV), an element of the Rabdoviridae family, has a single-stranded RNA genome, negative sense, not segmented. Its eleven kb genome has five genes which encode five structural proteins of the virus; the nucleocapsid protein (N); which is required in stoichiometric amounts for encapsidation of the replicated RNA; the phosphoprotein (P), which is a cofactor of RNA polymerase dependent on RNA (L); the matrix protein (M) and the binding glycoprotein (G) (eg, Gallione et al., 1981, Rose and Gallione, 1981; Rose and Schubert, .1987 and Schubert et al., 1985; US Patent 6,033,886; United States 6,168,943). VSV is a virus of arthropod origin that can be transmitted to a variety of mammalian hosts, most commonly cattle, horses, pigs and rodents. Human VSV infection is not common, and in general, it is either asymptomatic or characterized by symptoms similar to mild influenza that resolve in three to eight days without complications. Because VSV is not considered a human pathogen, and pre-existing immunity to VSV is rare in the human population, the development of VSV derived vectors has been a focus in areas such as immunogenic compositions and gene therapy. For example, studies have established that VSV can serve as a highly effective vector for immunogenic compositions, expressing influenza virus hemagglutinin (Roberts et al., 1999), measles H virus protein (Schlereth et al., 2000 ) and HIV-1 env and gag proteins (Rose et al., 2001). Other features of the VSV that provide an attractive vector include: (a) the ability to replicate robustly in cell cultures; (b) the inability to either integrate into the host cell's DNA or undergo genetic recombination; (c) the existence of multiple serotypes, allowing the possibility for primer-reinforcement immunization strategies; (d) foreign genes of interest can be inserted into the VSV genome and abundantly expressed by the viral transcriptase; and (e) the development of a highly specialized system for the rescue of infectious viruses from a cDNA copy of the virus genome (U.S. Patent 6,033,886; U.S. Patent 6,168,943). Although there is little evidence of the neurological involvement of VSV during natural infection, animals (eg, primates, rodents, pedigree animals) that are inoculated intracerebrally (and in the case of rodents intranasally) with the native-type virus, the virus Native type that passes to the mouse brain or native virus adapted to the cell culture, can develop clinical signs of disease, and usually die two to eight days after inoculation. Because of these observations, and the need to produce a vector for immunogenic compositions for human use that have an exceptional safety profile, VSV vectors under development are tested in rigorous neuroviruletry models in small and primate animals. These tests are designed to detect any residual virulence in attenuated VSV vectors before consideration for advancement in human clinical trials. The attenuation of prototypic VSV vectors results from the accumulation of multiple nucleotide substitutions through the genome of the virus during the serial step in vitro and the synthesis and assembly of the genome cDNA. These mutations have pleiotropic effects that provide the least pathogenic virus in mice that the virus adapted to the laboratory from which it is derived (for example, see Roberts et al., 1998). The additional prototypic attenuated VSV vectors were also developed by truncation of the cytoplasmic limb region of the virus G protein, leading to VSV mutants that were defective in budding the plasma membrane of infected cells (Schnell et al., 1998). Currently known VSV vectors, putatively attenuated or not, have had unacceptable levels of residual virulence when tested in small animals and in neurovirulence models in non-human primates. The development of a VSV vector for uses such as a vector for immunogenic compositions, a gene therapy vector and the like, will require VSV vectors having minimal to detectable levels of pathogenicity in models of animal neurovirulence. Thus, there is currently a need in the art for viral vectors to identify attenuated, genetically modified VSV mutants that have significantly reduced (or eliminated) pathogenicity in animals.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates broadly to the synergistic attenuation of vesicular stomatitis virus (VSV). More particularly, the invention relates to the identification of combined mutation classes, which synergistically attenuate the pathogenicity of VSV vectors in mammals and immunogenic compositions thereof. In this way, in certain modalities, the invention is directed to a Genetically modified VSV comprising at least two different classes of mutations in its genome, wherein the two mutations synergistically attenuate the pathogenicity of VSV. In a particular embodiment, the pathogenicity of VSV is also defined as neurovirulence. In another embodiment, the classes of mutations are a temperature-sensitive mutation (ts), a mutation site, a gene-mixing mutation, a G-trunk mutation, a mutation of the cytopathic M gene, an ambisense RNA mutation, a mutation of the truncated G gene, an insertion mutation of the G gene and a deletion mutation of the gene. In a particular embodiment, the two VSV mutations are a truncated G gene mutation (subsequently "G (Ct)") and a N gene mixing mutation (that is, the N gene is moved away from its first promoter position). -nearly 3 'of native type, to a more distal position in the order of the VSV gene). In another embodiment, the VSV G protein encoded by the truncated G gene has a deletion in at least twenty carboxy-terminal amino acids (subsequently, ".G (Ct-g)." In yet another embodiment, the VSV G protein encoded by the truncated G gene has a deletion of at least twenty-eight carboxy-terminal amino acids (subsequently, "G (Ct-i)." In still another embodiment, the N gene of the VSV is mixed to 3'-PNMGL-5 'or 3-PMNGL-5 ', in relation to the native VSV genome 3'-NPMGL-5', where N is the gene encoding the nucleocapsid protein, P is the gene encoding the phosphoprotein, M is the gene encoding the matrix protein, G is the gene encoding the binding glycoprotein and L is the gene encoding the RNA-dependent RNA polymerase protein.In certain embodiments, the VSV comprises a mutated genome of 3'-PNMG ( Ct-i) L-5 ', 3'-PNMG (Ct-9) L-5', 3'-PMNG (Ct-1) L-5 'or 3'-PMNG (ct-9) -5', where N is the gene that encodes the protein of the nucleus Apsid, P is the gene encoding the phosphoprotein, M is the gene encoding the matrix protein, G (Ct-i) is the gene encoding the binding glycoprotein having a cytoplasmic limb region consisting of an amino acid, G (Ct-9) is the gene encoding the binding glycoprotein having a cytoplasmic limb region consisting of nine amino acids and L is the gene encoding the RNA-dependent RNA polymerase protein. In a particular embodiment, the genome of the mutated VSV is 3'-PMNG (Ct-i) L-5 '. In another particular embodiment, the genome of the mutated VSV is 3'-PNMG (Ct-i) L-5 '. In another embodiment, the VSV further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, a mutation site, an ambisense RNA mutation, a G trunk mutation, a G gene insertion, a deletion of gene or a non-cytopathic M gene mutation. In certain embodiments, the modified VSV injected intracranially in female 4-week-old Swiss-Webster mice has a 100-fold higher LD50 than the native-type VSV injected intracranially in 4-week-old female Swiss-Webster mice. In certain other modalities, the VSV injected intracranially in female 4-week-old Swiss-Webster mice has an LD50 1, 000 times higher than the native VSV injected intracranially in female Swiss-Webster mice of 4 weeks of age. In still other embodiments, the VSV injected intracranially in female 4-week-old Swiss-Webster mice has an LD50 10,000 times greater than the native-type VSV injected intracranially in female Swiss-Webster mice at 4 weeks of age. In still other modalities, VSV injected intracranially in 4-week-old female Swiss-Webster mice, has an LD50 100,000 fold higher than the native VSV injected intracranially in female Swiss-Webster mice of 4 weeks of age. In other embodiments of the invention, the two VSV mutations are a truncated G gene mutation and a non-cytopathic M gene mutation. In certain embodiments, the G protein encoded by the truncated G gene has a cytoplasmic limb domain consisting of an amino acid (G (Ct-i)) or a cytoplasmic limb domain consisting of nine amino acids (G (Ct-9) )) - In other modalities, the non-cytopathic mutation of the M gene (subsequently, "M (ncp)"), is a mutation from methionine to alanine at position 33 (M33A) and a mutation from methionine to alanine at position 51 (M51A) of the M protein. In a particular embodiment, the VSV comprises a mutated genome of 3'-NPM (ncp) G (cM) L-5 'or 3'-NPM (ncp) G (ct-9) L -5'. In another embodiment, the VSV further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, an ambisense RNA mutation, a gene blending mutation, a gene deletion mutation, an insert mutation of gene, an insertion mutation of G gene, a truncal G mutation or a point mutation. As discussed below in Section A.3, a ts mutation of any of the VSV G, M, N, P or L genes is a "mutation class" separate from the invention. Thus, in certain embodiments of the invention, the two VSV mutations are a mutation of the N ts gene (subsequently, "N (tS)") and a L ts gene mutation (subsequently "L (tS)"). In a particular embodiment, the VSV comprises a mutated genome of 3'-N (ts) PMGL (tS) -5 '. In certain other embodiments, the VSV further comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a gene-blending mutation, a G-stem mutation, a non-cytopathic M-gene mutation, a mutation. of ambisense RNA, a truncated G gene mutation, a G gene insertion mutation or a gene deletion mutation. In certain embodiments, the two VSV mutations are a truncal G mutation (subsequently, "G (troncai)") and a gene mutation. In other embodiments, the VSV also comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a ts mutation, a gene-blending mutation, a non-cytopathic M-gene mutation, an RNA mutation. ambisense, a truncated G gene mutation, a G gene insertion mutation or a gene deletion mutation. In another embodiment, the invention is directed to a genetically modified VSV vector comprising at least two different classes of mutations in its genome and at least one foreign RNA sequence as a separate transcriptional unit inserted in or replacing a region of the VSV genome not essential for replication, where the two mutations synergistically attenuate the pathogenicity of VSV. As defined below, a "foreign RNA" sequence is any polynucleotide sequence which is not endogenous to the native VSV genome. In a particular embodiment, the pathogenicity of the vector is further defined as neurovirulence. In certain other modalities, foreign RNA is defined as an open reading structure (ORF). In certain other embodiments, the classes of mutations are selected from the group consisting of a ts mutation, a point mutation, a gene-blending mutation, a G-core mutation, a non-cytopathic M-gene mutation, an antisense RNA mutation. , a truncated G gene mutation, a G gene insertion mutation and a gene deletion mutation. In a particular embodiment, the two mutations of the VSV vector are a mutation of the truncated G gene and a mutation of the N gene mix. In another embodiment, the G protein encoded by the truncated G gene has a deletion of at least twenty carboxy amino acids. -terminals or a deletion of at least twenty-eight carboxy-terminal amino acids. In certain other modalities, the VSV vector of the N gene is mixed to 3'-PNMGL-5 'or 3'-PMNGL-5', in relation to the native VSV genome 3'-NPMGL-5 '. In a particular embodiment, the VSV vector comprises a mutated genome of 3'-PNMG (ct-i) L-5 ', 3'-PMNG (ct-i) L-5' or 3'-PMNG (Ct-9) L-5 '. In a particular embodiment, the genome of the mutated vector is 3'-PMNG (Ct-i) L-5 '. In another embodiment, the genome of the mutated vector is 3'-PNMG (Ct-i) L-5 '. In yet other embodiments, the VSV vector further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, a point mutation, an ambisense RNA mutation, a G truncation mutation, a mutation insertion G gene, a gene deletion mutation or a non-cytopathic M gene mutation. In certain other embodiments, the VSV injected intracranially in 4-week-old Swiss-Webster female mice has a 100-fold higher LD50 than the native-type VSV injected intracranially in 4-week-old female Swiss-Webster mice. In yet other embodiments, the modified VSV injected intracranially in female 4-week-old Swiss-Webster mice has a LOD501,000 fold higher than the native VSV injected intracranially in 4 week old female Swiss-Webster mice. In yet other embodiments, VSV intracranially injected into 4-week-old female Swiss-Webster mice has an LD50 10,000 times greater than the native-type VSV injected intracranially in female 4-week-old Swiss-Webster mice. In another embodiment, the VSV injected intracranially in female 4-week-old Swiss-Webster mice has an LD50 100,000 fold greater than the native VSV injected intracranially in female Swiss-Webster mice at 4 weeks of age. In certain other embodiments, the foreign RNA inserted into or replacing a region of the VSV genome not essential for replication is selected from the group consisting of an HIV gene, an HTLV gene, a SIV gene, an RSV gene, a PIV gene, an HSV gene, a CMV gene, an Epstein-Barr virus gene, a Varicella-Zoster virus gene, a mumps virus gene, a measles virus gene, a virus gene influenza, a poliovirus gene, a rhinovirus gene, a hepatitis A virus gene, a hepatitis B virus gene, a hepatitis C virus gene, a Norwaik virus gene, a togavirus gene, an alphavirus gene , a rubella virus gene, a rabies virus gene, a Marburg virus gene, an Ebola virus gene, a papilloma virus gene, a polyoma virus gene, a metapneumovirus gene, a gene coronavirus, a Vibrio cholera gene, a Streptococcus pneumoniae gene, a Streptococcus pyogenes gene, a Streptococcus agalactiae gene, a Neisseria meningitidis gene, a Neisseria gonorrheae gene, a Corynebacteria diphtheria gene, a Clostridium tetani gene, a Bordetella pertussis gene, a Helicobacter pylori gene, a Haemophilus gene, a Chlamydia gene, an Escherichia coli gene , a cytokine gene, a T helper epitope, a CTL epitope, an adjuvant gene and a co-factor gene. In a particular embodiment, the foreign RNA is an HIV gene selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev and vpu. In a particular embodiment, the HIV gene is gag, wherein the gag gene is inserted into the VSV genome in position one or in the fifth position. In particular embodiments, the genome of the mutated VSV vector is 3'-gag? -PNMG (ct-i) L-5 ', 3'-gag? -PNMG (ct-9) L-5? S-gagi-PMNGtct -ijL-d ', 3, -gag? -PMNG (ct-9) L-5', 3'-PNMG (ct-1) L-gag5-5 \ 3'-PNMG (ct-9) L-gag5 -5 \ 3'- PNMG (ct-1) L-gag5-5 'or S'-PMNG ^ L-gags-d'. In another embodiment, the foreign RNA expresses a tumor-specific antigen or antigen associated with the tumor, by induction of a protective immune response against a tumor (eg, a malignant tumor). Such tumor-associated or tumor-specific antigens include, but are not limited to, carcinoma antigen KS bread 1/4; Ovarian carcinoma antigen (CA125); Prosthetic acid phosphate; prostate-specific antigen; antigen associated with p97 melanoma; Gp75 melanoma antigen; High molecular weight melanoma antigen and prostate specific membrane antigen. In certain other embodiments, the two mutations of the VSV vector are a G (Ct) mutation and an M (nCp) mutation. In certain embodiments, the G protein encoded by the truncated G gene has a cytoplasmic limb domain consisting of an amino acid (G (C)) OR a cytoplasmic limb domain consisting of nine amino acids (G (Ct-9)) . In still other embodiments, the mutation M (ncp) is a mutation of methionine to alanine at position 33 (M33A) and a mutation from methionine to alanine at position 51 (M51A) of the M protein. In a particular embodiment, the The mutated genome is S'-NPMfncpjGfct-ijL-d 'or 3'-NPM (ncp) G (Ct-9) L-5'. In another embodiment, the vector further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, a point mutation, a gene blending mutation, a core G mutation, an ambisense RNA mutation, a gene insertion mutation G and a gene deletion mutation. In certain embodiments, the VSV vector comprises an HIV gene selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev or vpu. In a particular embodiment, the HIV gene is gag, wherein the mutated genome is 3'-NPM (ncp) G (ct-i) L-gag5-5 ', or 3'-NPM (ncP) G (Ct- 9) L-gag5-5 '. In still other embodiments, the two mutations of the VSV vector are a mutation of the N (tS) gene and a mutation of the L (tS) gene. In a particular embodiment, the vector comprises a mutated genome of 3'-N (ts) PMGL (tS) -5 '. In other embodiments, the vector further comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a gene-blending mutation, a truncal G mutation, a non-cytopathic G-gene mutation, a mutation of Ambisense RNA, a truncated G gene mutation, a G gene insertion mutation, and a gene deletion mutation. In certain embodiments, the VSV vector comprises an HIV gene selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev or vpu. In a particular embodiment, the HIV gene is gag, wherein the mutated genome is 3'-gag? -N (ts) PMGL (tS) -5 'or 3'-N (tS) PMGL (tS) -gag5- 5'. As discussed below in Section A.1, insertion of a foreign nucleic acid sequence (eg, HIV gag) into the VSV genome at 3 'in any of the N, P, M, G genes or L, effectively results in a "gene-blending mutation". Thus, in certain embodiments, the two mutations of the VSV vector are G mutation (trunk) and a gene mixing mutation. In one embodiment, the genome of the truncated vector is 3'-gag? -NPMG (tr0ncai) L-5 '. In other embodiments, the VSV vector also comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a ts mutation, a gene blending mutation, a non-cytopathic M gene mutation, a mutation of ambisense RNA, a truncated G gene mutation, a G gene insertion mutation and a gene deletion mutation. In another embodiment, the invention is directed to an immunogenic composition comprising an immunogenic dose of a genetically modified VSV vector, comprising at least two different classes of mutations in its genome and at least one foreign RNA sequence as a transcriptional unit. a separate insertion in or replacing a region of the VSV genome not essential for replication, where the two mutations synergistically attenuate the pathogenicity of the VSV. In another embodiment, the mutation classes are selected from the group consisting of a ts mutation, a point mutation, a gene-blending mutation, a G-core mutation, a non-cytopathic M-gene mutation, an ambisense RNA mutation, a truncated G gene mutation, a G gene insertion mutation and a gene deletion mutation. In certain modalities, the two mutations are a mutation of the truncated G gene and a mutation of the N gene mix. In particular embodiments, the G protein encoded by the truncated G gene has a cytoplasmic limb domain consisting of an amino acid (G (Ct- i)) or a cytoplasmic limb domain consisting of nine amino acids (G (ct-g)). In still other embodiments, the N gene is mixed to 3'-PNMGL-5 'or 3'-PMNGL-5 \ in relation to the native VSV genome 3'-NPMGL-5'.

In certain embodiments, the VSV vector of the immunogenic composition comprises a mutated genome of 3'-PNMG (Ct-i) L-5 ', 3'-PNMG (Ct-9) L-5', 3'- PMNG (ct-i) L-5 'or 3'-PMNG (Ct-9) L-5'. In a particular embodiment, the genome of the mutated VSV is 3'-PMNG (Ct-i) L-5 '. In another particular embodiment, the mutated VSV genome of the immunogenic composition is 3'-PNMG (Ct-i) L-5 '. In another embodiment, the genome of the mutated vector is 3'-PNMG (Ct-i) L-5 '. In other embodiments, the VSV vector of the immunogenic composition further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, an ambisense RNA mutation, a point mutation, a G mutation, a mutation. of G gene insertion, a gene deletion mutant or a non-cytopathic M gene mutation. In certain other embodiments, the foreign RNA inserted into the genetically modified VSV vector of the immunogenic composition is selected from the group consisting of an HIV gene, an HTLV gene, a SIV gene, an RSV gene, a PIV gene, a HSV gene, a CMV gene, an Epstein-Barr virus gene, a Varicella-Zoster virus gene, a mumps virus gene, a measles virus gene, an influenza virus gene, a gene poliovirus, a rhinovirus gene, a hepatitis A virus gene, a hepatitis B virus gene, a hepatitis C virus gene, a Norwaik virus gene, a togavirus gene, an alphavirus gene, a gene rubella virus, a rabies virus gene, a Marburg virus gene, an Ebola virus gene, a papilloma virus gene, a polyoma virus gene, a metapneumovirus gene, a coronavirus gene, a gene of Vibrio cholera, a gene of Streptococcus pneumoniae, a gene of Streptococcus pyogenes, a gene of Streptococcus agalactiae, a Neisseria meningitidis gene, a Neisseria gonorrheae gene, a Corynebacteria diphtheria gene, a Clostridium tetani gene, a Bordetella pertussis gene, a Helicobacter pylori gene, a Haemophilüs gene, a Chlamydia gene, an Escherichia coli gene , a cytokine gene, a T helper epitope, a CTL epitope, an adjuvant gene and a co-factor gene. In a particular embodiment, the foreign RNA encodes an HIV protein selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev and vpu. In a particular embodiment, the HIV gene is gag, wherein the gag gene is inserted into the VSV genome in position one or in the fifth position of the genome. In other embodiments, the VSV vector of the immunogenic composition comprises a 3'-gag mutated genome, -PNMG (ct-1) L-5 \ 3'-gag? -PNMG (ct-9) L-5? '-gagi-PMNG ^ DL-d', 3'-gagr 3'-PNMG (ct-i) L-gag5-5 'or S'-PMNG ^ L-gags-d'. In certain other embodiments, the VSV vector of the immunogenic composition comprises a mutation G (Ct) and a mutation (ncp) - In another embodiment, the G protein encoded by the truncated G gene has a cytoplasmic limb domain consisting of an amino acid (G (Cn)) or a cytoplasmic limb domain consisting of nine amino acids (G (ct-9)) - In other embodiments, the M mutation (pCp), is a mutation from methionine to alanine at position 33 (M33A) and a methionine to alanine mutation at position 51 (M51A) of the M protein. In a particular embodiment, the immunogenic composition comprises a mutated VSV genome of 3'-NPM (nCp) G (Ct-i) L-5 'or 3'-NPM (nCp) G (ct-9) L-5'. In still other embodiments, the VSV vector of the immunogenic composition further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, a point mutation, a gene blending mutation, a G stem mutation, an ambisense RNA mutation, a G gene insertion mutation, and a gene deletion mutation. In still other embodiments, the foreign RNA inserted into the genetically modified VSV vector of the immunogenic composition is selected from the group consisting of an HIV gene, an HTLV gene, a SIV gene, an RSV gene, a PIV gene, a HSV gene, a CMV gene, an Epstein-Barr virus gene, a Varicella-Zoster virus gene, a mumps virus gene, a measles virus gene, an influenza virus gene, a poliovirus gene, a rhinovirus gene, a gene for hepatitis A virus, a hepatitis B virus gene, a hepatitis C virus gene, a Norwaik virus gene, a togavirus gene, an alphavirus gene, a rubella virus gene, a rabies virus gene, a Marburg virus gene, an Ebola virus gene, a papilloma virus gene, a polyoma virus gene, a metapneumovirus gene, a coronavirus gene, a Vibrio cholera gene, a gene of Streptococcus pneumoniae, a gene of Streptococcus pyogenes, a gene of Helicobacter pylori, a gene of Streptococcus agalactiae, a gene of Neisseria meningitidis, a gene of Neisseria gonorrheae, a gene of Corynebacteria diphtheria, a gene of Clostridium tetani, a Bordetella gene pertussis, a Haemophilus gene, a Chlamydia gene, an Escherichia coli gene, a gene encoding a cytokine, a gene encoding a T helper, a gene encoding a CTL epitope, a gene encoding an adjuvant and a gene encoding a co-factor. In certain embodiments, the HIV gene is selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev or vpu. In a particular embodiment, the HIV gene is gag, where the mutated genome is 3'-gag? -NPM (ncp) G (Ct-i) L-5 ', 3'-gag NPMtncp ct-gjL-d' , 3'-NPM (ncp) G (ct-i) L-gag5-5 'or 3'-NPM (ncp) G (ct-9) L-gag5-5'. In certain other embodiments, the immunogenic composition comprises a mutation of the N (tS) gene and a mutation of the L (tS) gene. In a particular embodiment, the immunogenic composition comprises a mutated VSV genome of 3'-N (tS) PMGL (ts) -5 '. In other embodiments, the immunogenic composition further comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a gene blending mutation, a truncal G mutation, a non-cytopathic M gene mutation, a mutation of ambisense RNA, a truncated G gene mutation, a G gene insertion mutation and a gene deletion mutation. In still other embodiments, the foreign RNA inserted into the genetically modified VSV vector of the immunogenic composition is selected from the group consisting of an HIV gene, an HTLV gene, a SIV gene, an RSV gene, a PIV gene, a HSV gene, a CMV gene, an Epstein-Barr virus gene, a Varicella-Zoster virus gene, a mumps virus gene, a measles virus gene, an influenza virus gene, a gene poliovirus, a rhinovirus gene, a hepatitis A virus gene, a hepatitis B virus gene, a hepatitis C virus gene, a Norwaik virus gene, a togavirus gene, an alphavirus gene, a gene rubella virus, a rabies virus gene, a Marburg virus gene, an Ebola virus gene, a papilloma virus gene, a polyoma virus gene, a metapneumovirus gene, a coronavirus gene, a gene of Vibrio cholera, a gene of Streptococcus pneumoniae, a gene of Streptococcus pyogenes, a gene of Helicobacter pylori, a gene of S treptococcus agalactiae, a Neisseria meningitidis gene, a Neisseria gonorrheae gene, a Corynebacteria diphtheria gene, a Clostridium tetani gene, a Bordetella pertussis gene, a Haemophilus gene, a Chlamydia gene, an Escherichia coli gene, a gene encoding a cytokine, a gene encoding a T helper, a gene encoding a CTL epitope, a gene encoding an adjuvant and a gene encoding a co-factor. In certain embodiments, the HIV gene is selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev or vpu. In a particular embodiment, the HIV gene is gag, where the mutated genome is 3'-gag? -N (tS) PMGL (ts) -5 'or 3'-N (ts) PMGL (ts) -gag5- 5'. In certain other embodiments, the immunogenic composition comprises a G mutation (troncai) and a gene mixing mutation. In a particular embodiment, the immunogenic composition comprises a mutated genome of 3'-gag? -NPMG (troncai) L-5 '. In other modalities, the immunogenic composition further comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a ts mutation, a gene blending mutation, a non-cytopathic M gene mutation, an ambisense RNA mutation, a truncated G gene mutation, a G gene insertion mutation and a gene deletion mutation. In still another embodiment, an immunogenic composition of the invention is administered by any conventional route selected from the group consisting of intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, oral, rectal, intranasal, buccal, vaginal and ex vivo. In another embodiment, the invention is directed to a method for immunizing a mammalian subject against HIV infection comprising, administering to the subject, an immunogenic dose of a genetically modified VSV vector comprising at least two different classes of mutations in its genome and at least one HIV RNA sequence as a separate transcriptional unit in or replacing a region of the VSV genome not essential for replication, wherein the two mutations synergistically attenuate the pathogenicity of the VSV and the HIV RNA encodes a selected antigen of the group consisting of gag, env, pol, vif, nef, tat, vpr, rev and vpu. In certain modalities, the VSV vector is 3'-gagr PNMG (ct-i) L-5 ', 3'-gag1-PNMG (ct-9) L-5', 3'-gag PNMG (ct-9) L-5 ', 3'-PNMG (ct-1) L-gag5-5 \ 3'-PNMG (ct-9) L-gag5-5', 3-PMNG (ct-i) L- 3'-gag 3'-NPM (ncp) G (ct-1) L-gag5-5 ', 3'-NPM (ncp) G (ct-9) L-gag5-5', 3'-gag NttsjPMGL ^ -d 'or 3'-N (ts) PMGL (ts) -gag5-5'. In certain other embodiments, the invention is directed to a method for immunizing a mammalian host against a bacterial infection comprising administering an immunogenic dose of a vector of the genetically modified VSV comprising (a) at least two different classes of mutations in its genome , mutations are selected from the group consisting of a ts mutation, a point mutation, a mutation mixing gene, a trunk mutation G, a mutation of non-cytopathic M gene mutation RNA ambisense, a gene mutation G truncated, a G gene insertion mutation and a gene deletion mutation, wherein the two mutations synergistically attenuate the pathogenicity of the VSV and (b) at least one foreign RNA sequence inserted into or replacing a region of the VSV genome not essential for replication, where the RNA encodes a bacterial protein selected from the group consisting of a Vibrio cholera protein, a protein oteína Streptococcus pneumoniae, a protein of Streptococcus pyogenes, a protein of Streptococcus agalactiae, a protein of Helicobacter pylori, a protein of Neisseria meningitidis protein Neisseria gonorrhoeae, a protein of Corynebacterium diphtheria, a protein of Clostridium tetani, protein Bordetella pertussis, a Haemophilus protein, a Chlamydia protein and an Escherichia coli protein. In a particular embodiment, the two mutations are a G (Ct) mutation and a N gene mixing mutation. In certain embodiments, the G protein encoded by the truncated G gene has a cytoplasmic limb domain consisting of an amino acid (G (Cn)) or a cytoplasmic limb domain consisting of nine amino acids G (Ct-9). In certain other embodiments, the N gene is mixed to 3'-PNMGL-5 'or 3'-PMNGL-5', relative to the native VSV genome 3'-NPMGL-5 '. In other embodiments, the mutated VSV genome is 3'-PNMG (C) L-5 ', 3'-PNMG (ct-9) L-5', 3-PMNG (ct-i) L-5 ', or 3'-PMNG (Ct-9) L-5 '. In a particular embodiment, the mutated genome is 3'-PMNG (ct-i) L-5 'or 3'-PNMG (Ct-i) L-5'. In other embodiments, the VSV further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, a point mutation, a mutation RNA ambisense a deletion mutation of the gene, a trunk mutation G a G gene insertion mutation, a G gene insertion mutation, or a non-cytopathic M gene mutation. In another embodiment, the invention is directed to a method for immunizing a mammalian host against viral infection comprising, administering an immunogenic dose of a genetically modified VSV vector comprising (a) at least two different classes of mutations in its genome, mutations are selected from the group consisting of a ts mutation, a point mutation, a mutation mixing gene, a trunk mutation G, a mutation of non-cytopathic M gene mutation RNA ambisense, a mutation of G gene truncated , a G gene insertion mutation and a gene deletion mutation, wherein the two mutations synergistically attenuate the paiogenicity of the VSV and (b) at least one foreign RNA sequence inserted into or replacing a region of the VSV genome does not essential for replication, wherein the RNA encodes a viral protein selected from the group consisting of an HIV protein, an HTLV protein, a SIV protein, or an RSV protein, a PIV protein, an HSV protein, a CMV protein, an Epstein-Barr virus protein, a Varicella-Zoster virus protein, a mumps virus protein, a measles virus protein, a influenza virus protein, a poliovirus protein, a rhinovirus protein, a protein of the hepatitis A virus, a protein of the hepatitis B virus, a protein of the hepatitis C virus, a protein of the Norwaik virus, a protein togavirus, an alphavirus protein, a protein of the rubella virus, a rabies virus protein, a Marburg virus protein, an Ebola virus protein, a papilloma virus protein, a polyoma virus protein, a metapneumovirus protein and a coronavirus protein. In a particular embodiment, the RNA is an HIV gene selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev or vpu. In certain modalities, the two mutations are a mutation G (ct) and an N gene mixing mutation. In a particular embodiment, the mutated VSV genome is 3'-PNMG (ct-1) L-5 ', 3'-PNMG (ct-9) L-5 \ 3'PNMG (ct-i) L-5 'or 3'-PMNG (Cl-9) L-5'. In another embodiment, the HIV gene is gag, where the gag gene is inserted into the VSV genome at position one or position five, where the mutated genome is 3'-gagrPNMG (cM) L-5 ' , 3'-gagrPNMG (Ct-9) L-5 \ S'-gag PMNGíct-DL-d ', 3'-gag PNMG (ct-9) L-5 \ 3'-PNMG (ct-i) L- gag5-d ', 3'-PNMG (ct-9) L-gag5-d \ 3-PMNG (CM) L-gag5-d', or 3'-PMNG (ct-9) L-gag5-d \ In another embodiment, the VSV further comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, a point mutation, an ambisense RNA mutation, a gene deletion mutation, a G truncation mutation, a mutation of G gene insertion, a gene insertion mutation or a non-cytopathic M gene mutation. In other embodiments of the method for immunizing a mammalian host against viral infection, the two mutations of the VSV are a mutation G (Ct) and a mutation M (nCp) - In a particular embodiment, the genome of the mutated VSV is 3'- NPM (ncP) G (ct-1) L-5 'or 3'-NPM (nCp) G. { ct.9) L-5 '. In another embodiment, the mutated VSV genome is 3'-gag? -NPM (pcp) G (ct-i) L-d ', 3'-gag 0 3'-NPM (ncp) G (Ct-i) L -gag5-5 'or 3'-NPM (ncp) G (ct-g) L-gag5-5 *. In another embodiment, the VSV genome also comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, a point mutation, a gene-blending mutation, a G-stem mutation, an ambisense RNA mutation. , a G gene insertion mutation and a gene deletion mutation. In other embodiments of the method for immunizing a mammalian host against viral infection, the two VSV mutations are a mutation of the N (tS) gene and a mutation of the L (te) gene. In a particular embodiment, the mutated VSV genome is 3'-N (ts) PMGL (tS) -d ', 3'-gagr 0 N (tS) PMGL (ts) -5' or 3'-N (ts) PMGL (ts) -gag5-d '. In another embodiment, the VSV genome also comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a gene-blending mutation, a G-stem mutation, a non-cytopathic M-gene mutation, a 2d ambisense RNA mutation, a truncated G gene mutation, a G gene insertion mutation and a gene deletion mutation. In other embodiments of the method for immunizing a mammalian host against viral infection, the two VSV mutations are a G mutation (tr0ncai) and a gene mixing mutation. In one embodiment, the mutated genome is 3'-gag -? - NPMG (troncai) L-d '. In another embodiment, the VSV genome also comprises a third class of mutation in its genome, wherein the mutation is a point mutation, a ts mutation, a gene blending mutation, a non-cytopathic M gene mutation, a mutation of ambisense RNA, a truncated G gene mutation, a G gene insertion mutation and a gene deletion mutation. Other features and advantages of the invention will be apparent from the following detailed description, preferred embodiments thereof and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the growth kinetics (pfu / ml against time) of native VSV (3'-NPMGL-5 '), VSV mutants mixed in N (3'-PNMGL-5' [N2], 3 ' -PMNGL-d '[N3] and 3'-PMGNL-5' [N4]), truncating VSV mutants of the cytoplasmic limb (ct) of the G protein (3'-NPMG (ct-9) L-5 '[CT9] and 3'-NPMG (ct-1) L-gagd-d' [CT1-GAGd]) and truncation mutants ct of G protein / mixed in N of the combined VSV (3'- PNMG (ct-i ) Ld '[N2CT1], 3'-PNMG (Ct-9) Ld' [N2CT9], 3'-PMNG (ct-1) Ld '[N3CT1] and [N3CT9]). The abbreviation "inside" shown in the legend of the inserted figure represents the Indiana strain of the VSV. Figure 2 is a comparison of the growth kinetics of the mixed VSV mutants N (3'-PNMGL-d \ 3'-PMNGL-d 'and 3'-PMGNL-5') relative to the native VSV ( 3'-NPMGL-d ') and the VSV mutant ct-1 of the G protein (S'-NPMGcct-gjL-gags-d'). Figure 3 shows a comparison of the growth ratios of the mutant ct-1 protein G / mixed N of the combined VSV (3'-PNMG (Ct-1) Ld 'and 3'-PMNG (Ct-i) L- d '), in relation to the native VSV (3'-NPMGL-d') and a mutant of the VSV ct-1 of the G protein (3'-NPMG (ct-i) L-gag5-d ').

DETAILED DESCRIPTION OF THE INVENTION The invention described below addresses a need in the art for vesicular stomatitis virus (VSV) vectors that have significantly reduced pathogenicity in mammals, particularly attenuated neuropathogenicity, as revealed in models of animal neurovirulence. As described above, VSV has many characteristics which make an attractive vector for immunogenic compositions and / or gene therapy. For example, human VSV infection is not common and is either asymptomatic or characterized by symptoms similar to mild influenza that resolves in three to eight days without complications, and as such, VSV is not considered a human pathogen. Other features of the VSV that provide an attractive vector include: (a) the ability to replicate robustly in cell cultures; (b) the inability to either integrate into the DNA of the host cell or undergo genetic recombination; (c) the existence of multiple serotypes, allowing the possibility for primer-reinforcement immunization strategies; (d) foreign genes of interest can be inserted into the VSV genome and abundantly expressed by the viral transcriptase; (e) the development of a highly specialized system for the rescue of infectious viruses from a cDNA copy of the virus genome (US Patent 6,033,886; US Patent 6,168,943) and (f) the pre-existing immunity to VSV in the human population. it is not frequent. An early class of attenuated VSV vectors described in the art refers to temperature sensitive mutants (ts), where the ts mutants fail to produce virions at a restrictive temperature. For example, several ts mutants of VSV are known in the art (for example, see Holloway et al., 1970; Pringle et al., 1971; Evans et al., 1979; Pringle et al., 1981; Morita et al. , 1987, Gopalakrishna and Lenard, 198d). In addition, additional classes of attenuated VSV mutants have also been described in the art and include truncated cytoplasmic (ct) extremity mutations of the VSV G protein (Schnell et al., 1998), gene mixing mutations (or rearrangement). of the order of the gene) (Wertz et al., 1998; Ball et al., 1999; Flanagan et al., 2001; United States Patent 6, d96, d29), G trunking mutations (Jeetendra et al., 2003; , 2002; Robinson and Whitt, 2000), mutations of the non-cytopathic M protein (Jayakar et al., 2000; Jayakar and Whitt, 2002) and ambisense RNA mutations (Finke and Conzelmann, 1997; d Finke and Conzelmann 1999). However, as stated above, the attenuated VSV vectors currently available retain residual virulence when tested in animal models, and as such, are probably not candidate vectors for advancement in human clinical trials. As set forth in detail herein, the present invention relates to unexpected and surprising observations that combinations of two or more classes of known attenuating mutation (gene mixing mutations, G protein insertion and truncation mutations, mutations ts and other point mutations, non-cytopathic M d gene mutations, G-stem mutations, ambisense RNA mutations, gene deletion mutations and the like), have a synergistic effect (as opposed to an additive effect) on the level resulting from the attenuation of the pathogenicity achieved. For example, it is demonstrated here that the VSV G protein truncation mutants, when combined with mutants of the mixed N gene, exert a synergistic attenuation of VSV growth (Example 2) and neurovirulence (Example 3). In addition, certain embodiments of the present invention are directed to combinations of other kinds of mutations, which also have a synergistic effect on VSV attenuation. Such cases include, but are not limited to: ts mutations, point mutations, gene-blending mutations (including mixtures of N, P, M, G and L gene), G-stem mutations, G gene insertions, non-cytopathic M gene mutations, truncated G gene mutations (for example, a mutant ct), ambisense RNA mutations and gene deletion mutations. Thus, in certain embodiments, the invention is directed to a genetically modified VSV vector comprising at least two different classes of mutations in its genome and at least one sequence of 0 foreign RNA as a separate transcriptional unit inserted in or that it replaces a region of the non-essential VSV genome for replication, where the two mutations synergistically attenuate the pathogenicity of VSV. In certain other embodiments, the invention is directed to immunogenic compositions comprising a genetically modified VSV vector comprising at least two different classes of mutations in its genome and at least one foreign RNA sequence as a separate transcriptional unit inserted in or which replaces a non-essential VSV genome region for replication, where the two mutations synergistically attenuate the pathogenicity of VSV.

A. Mutation classes of vesicular stomatitis virus As stated above, a genetically modified VSV vector of the invention comprises at least two different classes of mutations in its genome. As defined below, the terms "mutation class", "mutation classes", or "mutation classes", are used interchangeably and refer to mutations known in the art, when used individually, to d attenuate the VSV. For example, a "mutation class" of the invention includes, but is not limited to, a VSV temperature-sensitive N gene mutation, "N (ts)"), a temperature-sensitive L gene mutation. (subsequently, "L (ts)"), a point mutation, a backbone G mutation (subsequently, "G (backbone)"), a non-cytopathic M gene mutation (subsequently, "M (ncp)"), 0 a gene rearrangement or mutation mutation, a truncated G gene mutation (subsequently, "G (Ct)"), an ambisense RNA mutation, a G gene insertion mutation, a gene deletion mutation and the like. As defined below, a "mutation" includes mutations known in the art as insertions, deletions, substitutions, gene rearrangements or mixed modifications. As defined below, the term "synergistic" attenuation refers to a level of VSV attenuation which is greater than the additive. For example, a synergistic attenuation of the VSV according to the present invention, comprises combining at least two classes of mutations in the same VSV genome, thereby, resulting in a reduction of the VSV pathogenicity much greater than a level of attenuation additive observed for each mutation class of the VSV alone. Thus, in certain embodiments, a synergistic attenuation of the VSV is defined as an LD50 at least greater than the level of additive attenuation observed for each mutation class alone (ie, the sum of the two classes of mutations), where the attenuation levels (ie, LD50) are determined in a small animal neurovirulence model, d By means of a non-limiting example, if equation (1) describes an "additive attenuation" of the VSV: (1)? a D50 +? b | _D50 = XLD5O; where? aLDso is the LD50 of a VSV that has a first mutation class in its genbma,? b? _D5o is the LDdO of a VSV that has a 0 second class of mutation in its genome and? XLDSO is the sum of? to Dso and? b [_D5o; then, a "synergistic attenuation" of the invention, having an LD5o at least greater than the level of additive attenuation observed for each mutation class alone, is described by equation (2): (2)? a, bLD5o > (? a D50 +? bLD5?); d where? a, b D5o is the LD50 of a VSV that has a combination of two classes of mutation in its genome,? a? _D5o is the LD55 of a VSV that has a first mutation class in its genome and? bLDso is the LD50 of a VSV that has a second class of mutation in its genome. Thus, in certain embodiments, the synergy of VSV attenuation (ie, two classes of mutation in the same VSV genome) is described in relation to the LD50 of two VSV constructs (each VSV construct has one class of single mutation in its genome), where the synergistic attenuation of VSV having two classes of mutation in its genome, is defined as an LD50 at least greater than the additive LD50 of the two VSV constructs that have a unique mutation class in its genome I (for example, see LD50 values of VSV in Table 7). In certain other embodiments, the synergy of d VSV attenuation is described in relation to the LD50 of a native VSV. Thus, in one embodiment, a synergistic attenuation of the VSV is defined as an LD50 that is at least, greater than the LD50 of I VSV of native type, wherein the LD50 is determined in a model of animal neurovirulence. In one embodiment, a synergistic attenuation of the VSV is defined as an LD50 that 0 is at least 10 times greater than the LD 0 of the native VSV, where the LD50 is determined in a model of animal neurovirulence. In another embodiment, a synergistic attenuation of the VSV is defined as an LD50 that is at least 100 times higher than the LD of the native VSV, where the LD50 is determined in an animal neurovirulence model. In another embodiment, a synergistic attenuation of the VSV is defined as an LD50 that is at least 1, 000 times greater than the LD50 of the native VSV, where the LD50 is determined in an animal neurovirulence model. In still other embodiments, a synergistic attenuation of the VSV is defined as an LD50 that is at least 10,000 times greater than the LD50 of the native VSV, where 0 the LD50 is determined in an animal neurovirulence model. In certain other embodiments, a synergistic attenuation of the VSV is defined as an LD50 that is at least 100,000 times greater than the LD50 of the native VSV, where the LD50 is determined in a model of animal neurovirulence. The determination of a lethal dose at dO% (LD5o) for a particular VSV vector is readily determined by a person skilled in the art, using known test methods and animal models (e.g., see Example 1). Thus, in certain embodiments, the invention is directed to a Gene-modified VSV comprising at least two different classes of mutations set forth below. 1. Gene 0 Mixing Mutations In certain embodiments, a genetically modified VSV of the invention comprises a gene mixing mutation in its genome. As defined herein, the terms "gene blending", "mixed gene", "mixed", "mixing", "gene rearrangement" and "gene translocation", are used interchangeably, and refer to a change d (mutation) in the genome order of the native VSV. As defined herein, a native VSV genome has the following gene order: 3'-NPMGL-d '. It is known in the art that the position of a VSV gene with respect to the 3 'promoter determines the level of expression and attenuation virus 0 (US Patent 6, d96, d29, and Wertz et al., 1998, each specifically incorporated in this document by reference). The nucleotide sequences encoding the VSV G, M, N, P and L proteins are known in the art (Rose and Gallione, 1981, Gallione et al., 1981). For example, U.S. Patent 6, d96, d29, describes gene blending mutations in which, the gene for N protein is translocated (mixed) from its first native-like proximal promoter position to successively more distal positions in the genome (for example, 3'-d PNMGL-d ', 3'-PMNGL-d', 3'-PMNGL-d \ referred to as N2, N3 and N4, respectively). Thus, in certain embodiments, a genetically modified VSV comprises a mutation of gene mix in its genome. In a mutation class, in a particular embodiment, a genetically modified VSV comprises a gene mixing mutation that 0 comprises a translocation of the N gene (e.g., 3'-PNMGL-5 'or 3'-PMNGL-d' ). It should be noted here that the insertion of a foreign nucleic acid sequence (eg gag of the IVH) into the 3 'VSV genome to any of the N, P, M, G or L genes, effectively results in a "gene blending mutation", as defined above. For example, when the HIV gag gene is inserted into the VSV genome at position one (for example, 3'-gagrNPMGL-d '), the N, P, M, G and L genes are each, moved from their native-type positions to more distal positions in the genome. Thus, in certain embodiments of the invention, a gene mixing mutation 0 includes the insertion of a foreign nucleic acid sequence into the 3 'VSV genome into any of the N, P, M, G or L genes ( for example, 3'-gag, -NPMGL-d \ 3'-N-gag2-PMGL-d ', 3'-NP-gag3-MGL-d', etc.). 3d 2. Insertion of protein g and truncation mutants In certain other embodiments, a genetically modified VSV of the invention comprises a mutated G gene, wherein the encoded G protein is truncated in its cytoplasmic domain (carboxy terminus), also referred to as the "cytoplasmic limb region" of the G protein. It is known in the art that mutations of the G gene which truncate the carboxy terminus of the cytoplasmic domain, influence the sprouting of VSV and attenuate the production of the virus (Schnell et al. , 1998; Roberts et al., 1999). The cytoplasmic I domain of the native type VSV G protein comprises twenty-nine 0 amino acids (RVGIHLCIKLKHTKKRQIYTDIEMNRLGK-COOH; SEQ ID NO: 1) - In certain modalities, a truncated VSV gene from the Invention, encodes a G protein in which the last twenty-eight carboxy-terminal amino acid residues of the cytoplasmic domain are deleted d (retaining only arginine from the cytoplasmic domain of the native twenty-nine amino acid type of SEQ ID NO: 1). In certain other embodiments, a truncated VSV G gene of the invention encodes a G protein in which the last twenty carboxy-terminal amino acid residues of the cytoplasmic domain are deleted (relative to the native cytoplasmic domain of twenty-nine amino acids of the cytoplasmic domain). SEQ ID NO: 1). In certain other modalities, a truncated VSV gene of the invention, encodes a G protein comprising a single amino acid in its cytoplasmic domain (cytoplasmic limb region), wherein the single amino acid is any naturally occurring amino acid. In yet other embodiments, a truncated VSV G gene of the invention encodes a G protein comprising nine amino acids in its cytoplasmic domain (cytoplasmic limb region), wherein the nine amino acids are any of the naturally occurring amino acids. In certain other embodiments, a mutated VSV gene of the invention encodes a G protein that contains an insert representing a foreign epitope. Such mutants are known in the art (for example, see Schlehuber and Rose, 2003). As defined herein, a mutant of the G gene encoding a G protein in which the last twenty-eight carboxy-terminal amino acid residues of the cytoplasmic domain are deleted, relative to the native-type sequence of SEQ ID NO: 1 , is designated "G (Ct-i)", wherein the cytoplasmic domain of G (Ct-i) has an amino acid sequence of (R-COOH). As defined herein, a mutant of the G gene encoding a G protein in which the last twenty carboxy-terminal amino acid residues of the cytoplasmic domain are deleted, relative to the native type sequence of SEQ ID NO: 1 , is designated "G (Ct-9)", where the cytoplasmic domain of G (ct-9) has an amino acid sequence of (RVGIHLCIK- COOH); SEQ ID NO: 2). Thus, in certain embodiments of the invention a genetically modified VSV of the invention comprises a mutated G gene, wherein the encoded G protein is a G (ct-i) or G (Ct-9). 3. Knot mutations sensitive to temperature and others. A "temperature sensitive" ("ts") mutation of the VSV, as defined below, is a mutation in the VSV genome which restricts the growth of the VSV at an impermissible temperature. For example, a ts mutant of the VSV of the invention grows normally and at high titration at the permissible temperature (eg, 31 ° C), but its growth or reproduction is restricted to non-permissive temperatures (eg, 37 ° C or 39 ° C). The generation of ts mutants by site-directed mutagenesis and chemistry is well known in the art (for example, see Pringle, 1970; Li et al., 1988); and numerous ts mutants have been characterized and described (for example, see Flamand and Pringle, 1971, Flamand and Bishop, 1973, Printz and Wagner, 1971, Gopalakrishna and Lenard, 198d, Pringle et al., 1981; Morita et al., 1987; Li et al., 1988; Rabinowitz et al., 1977; Lundh et al., 1988; Dal Canto ei al., 1976; Rabinowitz et al., 1976). In certain embodiments, a genetically modified VSV of the invention comprises a ts mutation in its genome, wherein the ts mutation is one or more mutations of a nucleic acid sequence encoding the G, M, N, P or L protein As defined herein, a ts mutation of any of the VSV G, M, N, P or L genes is a separate "mutation class" of the invention. For example, in certain embodiments of the invention, a genetically modified VSV comprises at least two different classes of mutations in its genome (wherein the two mutations synergistically attenuate the pathogenicity of the VSV), comprising one or more mutation (s) of the gene N ts (subsequently "N (t S)") as a first mutation class and one or more mutation (s) of the L ts gene (subsequently, "L (t S)") as a second mutation class. As a non-limiting example, a genetically modified VSV comprises a genome such as 3'-N (tS) PMGL (ts) -d 'comprising two classes of mutations (i.e., (1) a mutation of the N gene (tS) ) and (2) a mutation of the L (tS) gene and a genetically modified VSV comprising a genome such as 3'-gag? -N (tS) PMGL (tS) -d 'comprising three classes of mutations (ie to say, (1) a mutation of the N (tS) gene, (2) a mutation of the L (ts) gene and (3) by gagí insertion, a gene mixing mutation). In certain other embodiments, a genetically modified VSV of the invention comprises a point mutation in its genome, wherein the point mutation is one or more mutations of a nucleic acid sequence encoding the G, M, N protein, P or L, wherein the mutation confers an attenuating phenotype such as cold adaptation, decreased fusion or cytopathogenic efficiency (for example, see Fredericksen and Whitt, 1998, Ahmed and Lyles, 1997). For example, Fredericksen and Whitt (1998), describe three attenuating point mutations of the G gene (eg, D137-L, E139-L or DE-SS), which have a pH threshold changed by fusion activity. Ahmed and Lyles (1997) describe a mutation point of attenuation of the M gene (N1 63D), which was highly defective in the inhibition of host gene expression and was changed more rapidly than the native type M protein. Thus, in certain embodiments, a genetically modified VSV of the invention comprises one or more point mutations in its genome. 4. Mutations of the non-cytopathic M gene In certain other embodiments, a genetically modified VSV d of the invention comprises a non-cytopathic mutation in the M gene. The M gene of the VSV (Indiana serotype) encodes an M protein of 229 amino acids (matrix) , wherein the first thirty amino acids of the NH2 term, comprise a PPPY (PY) portion rich in proline (Harty et al., 1999). The PY portion of a VSV M protein is located at the 0 amino acid positions 24-27 in serotypes both Indiana (Genbank Access Number X0442) and New Jersey (Genbank Access Number M14553). It was shown by Jayakar and al. (2000), that the mutations in the PY portion (for example, APPY, AAPY, PPAY, APPA, AAPA and PPPA), reduce the yield of the virus by blocking a late stage in the sprouting of the virus. Thus, in certain embodiments, a genetically modified VSV of the invention comprises a non-cytopathic mutation in the M gene, wherein the mutation is in the PPPY portion of the encoded M protein. It has been recently reported that M mRNA also encodes two additional proteins, referred to as M2 and M3 (Jayakar and Whitt, 2002). The M2 and M3 proteins are synthesized from the methionines downstream in the same reading structure that encodes the M protein of 229 amino acids (referred to as M1), and lacks the first thirty two (M2 protein) or fifty (M protein) amino acids of the M1 protein. It has been observed that cells infected with a recombinant VSV expressing the M protein, but not M2 and M3, have a delayed onset of cytopathic effect (in certain cell types), which still produces a normal virus yield. Thus, in certain embodiments, a genetically modified VSV of the invention comprises a non-cytopathic mutation in the M gene, wherein the mutation of the M gene results in a virus that does not express the M2 or M3 protein (see for example , Jayakar and Whitt, 2002). Also contemplated herein are amino acid mutations (e.g., deletions, substitutions, insertions, etc.) in the PSAP (PS) portion of the M protein described by Irie et al. (2004). d. Trunk Mutations G In certain embodiments, a genetically modified VSV of the invention comprises a mutation in the G gene, wherein the encoded G d protein has a mutation in the membrane-proximal trunk region of the G protein ectodomain, referred to as a backbone protein G. The backbone region G comprises amino acid residues 421 to 462 of the G protein. Recent studies have demonstrated the attenuation of VSV via insertion and / or deletion mutations (eg, truncation), in the backbone G of the G protein (Robinson and Whitt, 2000; Jeetendra ei a /., 2002; Jeetendra ei al, 2003). Thus, in certain embodiments, a genetically modified VSV comprises an insertion, deletion, truncal replacement G, or combination thereof. In a particular embodiment, a genetically modified VSV vector of the invention comprises a G trunk mutation (and immunogenic compositions thereof), comprising a genome of 3'-gagrNPMG (troncai) L-5 '. 6. Ambisense RNA Mutations In certain embodiments, a genetically modified VSV of the invention comprises an ambisense RNA mutation, in which the d 'antigenome promoter (AGP) is replaced with a copy of the 3' genome promoter (GP ). The AGV d 'of VSV, as well as other non-segmented, negative-strand RNA viruses, act as a strong replication promoter, while the 3' of GP acts as a transcription promoter and a weak replication promoter. In the normal course of VSV infection, there is a 3 to 4 fold predominance of genome copies over copies of the antigenome; This relationship is even higher for rabies virus, another element of the Rhabdovirus family (Finke and Conzelmann, 1999). Previous work with rabies virus demonstrated that replacing the 5 'AGP with a copy of the GP (known as an ambisense RNA mutation), leads to equal levels of genome and antigenome RNA copies with infected cells. In addition, a foreign gene was expressed from the GP copy placed at the d 'end of the genome. When serially passed into cultured cells, the rabies virus contains the ambisense RNA mutation, consistently replicating 10 to 1 d times lower titers than a recombinant native-type rabies virus (Finke and Conzelmann, 1997). Such mutations are used in VSV vectors to attenuate both the replication of the virus and the expression of foreign genes. Thus, in certain embodiments, a genetically modified VSV comprises a mutation of ambisense RNA. d 7. Gene deletions In certain other embodiments, a genetically modified VSV of the invention comprises a virus in which a VSV gene (such as G or M) is deleted from the genome. For example, Roberts went to. (1999), describes a VSV vector in which the complete gene coding for the G protein is deleted (? G) and substituted with the influenza hemagglutinin protein (HA), where the VSV vector (? G-HA), demonstrated attenuated pathogenesis.

B. Recombinant Vectors of Vesicular Stomatitis Virus d In certain embodiments, the invention provides a vector of the Genetically modified VSV (recombinant) comprising at least two different classes of mutations in its genome and at least one foreign RNA sequence inserted as a separate transcriptional unit or replacing a region of the VSV genome not essential for replication. Methods for producing recombinant RNA viruses are referred to in the art as "recapture" or "reverse genetics" methods. Exemplary rescue methods for VSV are described in U.S. Patent 6,033,886, U.S. Patent 6,596,529 and WO 2004/113617, each incorporated herein by reference. The transcription and replication of RNA viral genomes, unsegmented, single strand, negative sense, are achieved through the enzymatic activity of a multimeric protein complex that acts in the core of the ribonucleoprotein (nucleocapsid). The naked genomic RNA, it can not serve as a template. Instead, these genomic sequences are recognized only when they are completely encapsidated by the N protein in the nucleocapsid structure. It is only in such a context that genomic and antigenomic terminal promoter sequences are recognized for initiating replication and transcriptional trajectories. An equivalent cloned DNA of the VSV genome is placed between a suitable DNA-dependent RNA polymerase promoter (e.g., the T7 RNA polymerase promoter), and a self-splitting ribozyme sequence (e.g., delta ribozyme). of hepatitis), which is inserted into a suitable transcription vector (for example, the propagating bacterial plasmid). This transcription vector provides the easily manipulated DNA template from which the RNA polymerase (eg, T7 RNA polymerase) can faithfully transcribe a single-stranded RNA copy of the antigenome (or genome) of the VSV, with the term precise, or almost accurate d 'and 3'. The orientation of the copy of the genomic DNA and the flanking promoter and the ribozyme sequences determine whether the RNA equivalents of the genome or antigenome are transcribed. Also required for the rescue of new VSV progeny, are the VSV-specific trans-acting carrier proteins needed to encapsulate the RNA transcripts of the single-stranded VSV genome or antigenome in nude, functional nucleocapsid templates: viral nucleocapsid (N), the phosphoprotein associated with the polymerase (P) and the polymerase protein (L). d These proteins comprise the RNA polymerase dependent on the active viral RNA, which must couple this nucleocapsid template to achieve transcription and replication. Thus, a genetically modified and attenuated VSV of the invention, comprising at least two different classes of mutations in its genome (e.g., see Section A), is produced in accordance with the methods of rescue known in the art. . For example, a genetically modified VSV vector comprising at least two different classes of mutations in its genome is generated using (1) a transcription vector comprising an isolated nucleic acid molecule, which comprises d a polynucleotide sequence that encodes a VSV genome or antigenome and (2) at least one expression vector which comprises at least one isolated nucleic acid molecule encoding the N, P and L transducing proteins necessary for packaging, transcription and replication; in a host cell under conditions sufficient to allow the co-expression of these vectors and the production of the recombinant VSV. Any strain or serotype of the VSV may be used in accordance with the present invention, which includes but is not limited to Indiana VSV; VSV of New Jersey, VSV of Chandipura, VSV of Isfahan, VSV of San Juan, VSV of Glasgow and the like. 4d In addition to the polynucleotide sequences encoding attenuated forms of the VSV, the polynucleotide sequence can also encode one or more heterologous (or foreign) sequences or open reading structures (ORF) (see for example, Section C). The heterologous polynucleotide sequences may vary as desired, and include but are not limited to, a co-factor, a cytokine (such as an interleukin), a helper epitope T, a CTL epitope, a restriction marker, an adjuvant, or a protein from a different microbial pathogen (eg, virus, bacteria, parasite or fungus), proteins especially capable of eliciting a desirable immune response. In certain embodiments, a heterologous ORF contains an HIV gene (eg, gag, env, pol, vif, nef, tat, vpr, rev or vpu). In a particular embodiment, the HIV gene is gag, wherein the gag gene is inserted into the VSV genome at position one (3'-gagrNPMGL-5 ') or at position five (3'-NPMG-gag - L5 '). The heterologous polynucleotide is also used to provide agents which are used for gene therapy. In another embodiment, the heterologous polynucleotide sequence further encodes a cytokine, such as an interleukin-12, which are selected to enhance the prophylactic or therapeutic characteristics of the recombinant VSV. In certain embodiments, a genetically modified and attenuated VSV of the invention is mutated by conventional means, such as chemical mutagenesis. For example, during the growth of the virus in cell cultures, a chemical mutagen is added, followed by: (a) selection of the virus that has been subjected to steps at suboptimal temperature to select mutations adapted to cold and / or temperature sensitive, (b) identification of the mutant virus that produces small plaques in the cell culture, and / (c) passage through heterologous hosts to select the host interval mutations. In other embodiments, attenuation mutations d comprise making predetermined mutations using site-directed mutagenesis (e.g., see Section A) and then retrieving viruses containing these mutations. As set forth above, a genetically modified VSV of the invention comprises at least two kinds of mutation in its genome. In certain embodiments, one or more mutation classes in addition to 0 comprise multiple mutations, such as a trunk mutation class G having a double mutation (eg, deletion, insertion, substitution, etc.), a triple mutation and the like. These attenuated VSV vectors are then selected for attenuation of their virulence in an animal model (for example, see Example 1 and Example 3). d Typical circumstances (although not necessarily exclusive) for rescue include, a thousand appropriate mammalian cells in which, the T7 polymerase is present to drive the transcription of the single-stranded antigenomic (or genomic) RNA, of the transcription vector containing the viral genomic cDNA. Either transcriptionally or briefly thereafter, this viral antigenome (or genome) RNA transcript is encapsidated into functional templates by the nucleocapsid protein and coupled by the required polymerase components concurrently produced from the co-transfected expression plasmids that encode the specific trans-acting proteins of the virus required. These events and processes lead to the prerequisite transcription of viral mRNAs, the replication and amplification of new genomes and, with it, the production of new progenies of VSV, that is, rescue. The transcription vector and the expression vector are typically plasmid vectors designed for expression in host cells. The expression vector which comprises at least one isolated nucleic acid molecule encoding the necessary trans-acting proteins for packaging, transcription and replication, expresses these proteins from the same expression vector or from at least two different vectors. These vectors are generally known from basic rescue methods and do not need to be altered for use in the improved methods of this invention. d Additional techniques to conduct virus rescue such as VSV are described in U.S. Patent 6,673,672 and Provisional U.S. Patent 60 / 477,389, which are hereby incorporated by reference. The host cells used in the rescue of the VSV are 0 those which allow the expression of the vectors of the necessary constituents for the production of the recombinant VSV. Such host cells can be selected from a prokaryotic cell or a eukaryotic cell, and preferably, a vertebrate cell. In general, host cells are derived from a human cell, such as a human embryonic kidney cell (e.g., 293). Vero cells, as well as many other cell types, are also used as host cells. The following are non-limiting examples of host cells: (1) d lines of human diploid primary cells (e.g., WI-38 and MCRd cells); (2) Monoclonal diploid cell line (e.g., Rhesus FRhL-Fetal lung cells); (3) Continuous Cuasi Primary Cell Line (eg, African Green Monkey Kidney cells AGMK); (4) human 293 cells and (d) other potential cell lines, such as CHO, MDCK 0 (Madin-Darby canine kidney), chicken embryo primary fibroblasts. In certain embodiments, a reagent that facilitates transfection is added to increase the uptake of DNA by the cells. Many of these reagents are known in the art (eg, calcium phosphate). Lipofectate (Life Technologies, Gaithersburg, MD) and Efecteno (Qiagen, Valencia CA), are common examples. Lipofectate and Efectene are both cationic lipids. They are both DNA coated and improve the absorption of DNA by the cells. The Lipofectato forms a liposome that surrounds the DNA while the Efecteno covers the DNA but does not form a liposome. The rescued attenuated VSV is then tested for its desired phenotype 0 (temperature sensitivity, cold adaptation, plate morphology and transcription and replication attenuation), first by in vitro means. Mutations are also tested using a minireplicon system wherein the required trans-acting encapsidation and polymerase activities are provided by vaccine helper or native viruses, or by plasmids expressing the different N, P and L genes harboring the gene-specific attenuation mutations. The attenuated VSV is also tested in vivo for synergistic attenuation in an animal neurovirulence model. For example, mouse and / or ferret models are established to detect neurovirulence. Briefly, groups of ten mice are injected intra-cranially (IC) with each of a scale of virus concentrations running through the anticipated LD50 dose (a dose that is lethal to 60% of animals). For example, IC inoculation with the virus at 102, 103, 104 and 105 pfu is used where the LD5o anticipated for the virus is in the escape of 103-104 pfu. The virus formulations are prepared by serial dilution of purified viruses stacked in PBS. The mice are then injected through the top of the skull with the requisite dose, in 60-100 μl of PBS. The animals are monitored daily for weight loss, morbidity and death. The LD50 for a virus vector is then calculated from the cumulative death of mice on a scale of tested concentrations.

C. Heterologous Nucleic Acid and Antigenic Sequences In certain embodiments, the invention provides sinergistically attenuated VSV 0 which further comprises a sequence of foreign RNA as a separate transcriptional unit inserted in or replacing a non-essential genome site for replication, in where the foreign RNA sequence (which is the negative sense) directs the dO production of a protein capable of being expressed in a host cell infected by the VSV. This recombinant genome is originally produced by inserting the foreign DNA encoding the protein into the VSV cDNA. In certain modalities, any DNA sequence which encodes an immunogenic antigen, which produces prophylactic or therapeutic immunity against a disease or disorder, when expressed as a fusion protein or non-fusion in a synergistically attenuated recombinant VSV of the invention, alone or in combination with other antigens expressed by the same or different VSV, it is isolated and incorporated into the VSV vector for use in the immunogenic compositions of the present invention. In certain embodiments, the expression of an antigen by a synergistically attenuated VSV induces an immune response against a pathogenic microorganism. For example, an antigen may exhibit the immunogenicity or antigenicity of an antigen found in bacteria, parasites, viruses or fungi, which are causative agents of diseases or disorders. In one embodiment, antigens that exhibit the antigenicity or immunogenicity of an antigen of a human pathogen or other antigens of interest are used. To determine the immunogenicity or antigenicity by detecting binding to the antibody, various immunoassays known in the art are used, including but not limited to competitive and non-competitive assay systems, using techniques such as radioimmunoassay, ELISA (immunosorbent assay linked to the enzyme), "interspersed" immunoassays, immunoradiometric assays, precipitation reactions by gene diffusion, immunodiffusion assays, in situ immunoassays (using colloidal gold, radioisotope labels or enzymes for example), western blotting, immunoprecipitation reactions, immunoassay assays, agglutination (eg, gene agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays and immunoelectrophoresis assays, neutralization assays, etc. In one embodiment, the antibody binding is measured by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by measuring the binding of an antibody or secondary reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art to detect binding in an immunoassay. In an embodiment for detecting immunogenicity, responses mediated by T cells are assayed by standard methods, eg, in vitro or in vivo cytotoxicity assays, tetramer assays, elispot assays, or in vivo delayed-type hypersensitivity assays. Bacteria and parasites that express epitopes (antigenic determinants) that are expressed by the synergistically attenuated VSV (where the foreign RNA directs the production of a parasite or bacterial antigen or a derivative thereof that contains an epitope thereof) include but are not limited to they are not limited to those listed in Table 1. 62 TABLE 1 Parasites and bacteria that express epitopes that can be expressed by VSV PARASITES plasmodium spp. elmeria spp. nematodes schist leshmania BACTERIA Vibrio cholerae Streptococcus pneumoniae Streptococcus agalactlae Neisseria meningitidis Neisseria gonorrheae Corynebacteria diphtheriae Clostridium tetani Bordetella pertussis Haemophilus spp. (for example, influenza) Chlamydia spp. Enterotoxigenic Escherichia coli Helicobacter pylori Mycobacteria In another embodiment, the antigen comprises an epitope of an antigen of a nematode, to protect against disorders caused by such worms. In another embodiment, any DNA sequence which encodes a Plasmodium epitope, which when expressed by a recombinant VSV, is immunogenic in a vertebrate host, is isolated for insertion in the VSV DNA (-) according to the present invention. The species of Plasmodium which serve as a source of DNA, include but not they limit to, the parasites of human malaria, P. falciparum, P. malariae, P. ovale, P. vivax and the parasites of animal malaria P. berghei, P. yoelii, P. 63 knowlesi, and P. cynomolgi. In yet another embodiment, the antigen comprises a peptide of the β-subunit of the cholera toxin. Viruses that express epitopes that are expressed by syngogically attenuated VSV (where foreign RNA directs the production of an antigen of the virus or a derivative thereof, comprising an epitope of the same), include but are not limited to, those listed in Table 2, which lists such viruses per family, for convenience purposes and without limitation.

TABLE 2 Viruses expressing epitopes that can be expressed by VSV I. Picornaviridae Enterovirus Poliovirus Coxsackievirus Echovirus Rhinovirus Hepatitis A Virus II. Caliciviridae Virus groups Norwaik lll. Togaviridae and Flaviviridae Togavirus (eg, Dengue virus) Flavivirus alphavirus (eg, Hepatitis C virus) Rubella virus IV. Coronaviridae Coronavirus V. Rhabdoviridae Rabies Virus VI. Filoviridae Virus Marburg Ebola Virus 64 TABLE 2 (CONTINUED) Viruses expressing epitopes that can be expressed by VSV Vile. Paramyxoviridae Influenza virus Mumps virus Sarapion virus Respiratory syncytial virus Metapneumovirus VIII. Orthomyxoviridae Orthomixovirus (for example, influenza virus) IX. Bunyaviridae Buniavirus X. Arenaviridae Arenavirus XI. Reoviridae Reovirus Rotavirus Orbivirus Xll. Retroviridae Human T cell leukemia virus type Human T cell leukemia virus type II Human immunodeficiency virus (e.g., type I and type II) Simian immunodeficiency virus Lentivirus Xlll. Papoviridae Poliomavirus Papilomavirus 5d TABLE 2 (CONTINUED) Viruses expressing epitopes that can be expressed by VSV XIV. Parvoviridae Parvovirus XV. Herpesviridae Herpes simplex virus Epstein-Barr virus Cytomegalovirus Varicella-Zoster virus Herpesvirus-6 human Herpesvirus-7 human Herpes virus Cercopithecine 1 (virus B) XVI. Poxviridae Poxvirus XVIII. Hepadnaviridae Hepatitis B Virus XIX. Adenoviridae In specific modalities, the antigen encoded by the foreign sequences that is expressed after infection of a host by the attenuated VSV, presents the antigenicity or immunogenicity of a hemagglutinin of the influenza virus; syncytial G virus glycoprotein human respiratory (G); measles virus haemagglutinin or glycoprotein gD of herpes simplex virus type 2.

Other antigens that are expressed by attenuated VSV include, but are not limited to, those that exhibit the antigenicity or immunogenicity of the following antigens: Poliovirus 1 VP1; envelope glycoproteins of HIV 1; antigen from the surface of Hepatitis B; Diphtheria toxin; Streptococcus epitope 24M, SpeA, SpeB, SpeC, or peptidase Cda; and gonococcal pilin. In other embodiments, the antigen expressed by the attenuated VSV exhibits the antigenicity or immunogenicity of pseudorabies gdO virus (gpD), pseudorabies virus II (gpB), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, glycoprotein 195 of transmissible gastroenteritis matrix protein TGE glycoprotein 38 swine rotavirus capsid protein porcine parvovirus, protective antigen Serpulina hydodysenteriae, glycoprotein 55 viral Diarrhea coil, hemagglutinin-neuraminidase virus disease Newclaste, hemagglutinin swine flu, or swine flu neuraminidase. In certain embodiments, an antigen expressed by the attenuated VSV exhibits the antigenicity or immunogenicity of an antigen derived from a canine or feline pathogen, including but not limited to, feline leukemia virus, canine distemper virus, canine adenovirus, parvovirus. canine and the like. In certain other modalities, the antigen expressed by the attenuated VSV has the antigenicity or immunogenicity of an antigen derived from Serpulina hyodysenteriae, Foot and Mouth Disease Virus, Hog cholera virus, swine influenza virus, swine influenza virus, African swine fever, Mycoplasma hiponeumoniae, infectious bovine rhinotracheitis (eg glycoprotein E virus IBR or 67 glycoprotein G), or infectious laryngotracheitis virus (e.g., glycoprotein G or gicoproteína I ILTV) . In another embodiment, the antigen has the antigenicity or immunogenicity of a La Crosse virus glycoprotein, neonatal calf diarrhea virus, Venezuelan equine encephalomyelitis virus, Punta Toro virus, murine leukemia virus or mouse mammary tumor virus. In other embodiments, the antigen exhibits the antigenicity or immunogenicity of an antigen of a human pathogen, including but not limited to, human herpesvirus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-virus. Barr, Varicella-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza virus, human immunodeficiency virus (type 1 and / or type 2), rabies virus, measles virus, human hepatitis B, hepatitis C virus, Plasmodium falciparum, and Bordetella pertussis. Potentially useful antigens or derivatives thereof for use as attenuated VSV expressed antigens are identified by several criteria, such as antigen involvement in the neutralization of an infectivity of the pathogen, type or group of specificity, recognition by immune cells or antiserum of the patient, and / or demonstration of protective effects of antiserum or antigen-specific immune cells. In another embodiment, the attenuated VSV foreign RNA directs the production of an antigen comprising an epitope, which when attenuated VSV is introduced into a desired host, induces an immune response that protects against a condition or disorder caused by an immune response. entity that contains the epitope. For example, the antigen may be a tumor-specific antigen or tumor-associated antigen, by induction of a protective immune response against a tumor (e.g., a malignant tumor), Such tumor-associated or tumor-specific antigens, include but are not are limited to, pancreatic antigen KS 1/4; Ovarian carcinoma antigen (CA126); prostatic acid phosphate; prostate-specific antigen; p97 antigen associated with melanoma; High molecular weight melanoma antigen and prostate specific membrane antigen. The foreign DNA encoding the antigen, which is inserted into a non-essential site of the attenuated VSV DNA, optionally further comprises a foreign DNA sequence encoding a cytokine capable of being expressed and stimulating an immune response in a host infected by the VSV attenuated. For example, such cytokines include but are not limited to, interleukins 1a, 1ß, 2, 4, d, 6, 7, 8, 10, 12, 13, 14, 16, 16, 17 and 18, interferon-a, interferon -β, interferon-α, granulocyte colony stimulation factor, granulocyte macrophage colony stimulation factor and tumor necrosis factors a and β. d. Immunogenic and pharmaceutical compositions In certain embodiments, the invention is directed to an immunogenic composition comprising an immunogenic dose of a genetically modified VSV vector comprising at least two different classes of 69 mutations in its genome and at least one foreign RNA sequence. inserted into or replacing a region of the VSV genome not essential for replication, where the two mutations synergistically attenuate the pathogenicity of VSV. d The synergistically attenuated VSV vectors of the invention are formulated for administration to a mammalian subject (eg, a human). Such compositions typically comprise the VSV vector and a pharmaceutically acceptable carrier. As used below, the language "pharmaceutically acceptable carrier" is intended to include any of all solvents, dispersion media, coatings, antibacterial and antifungal reagents, antisickers, and the like, compatible with the pharmaceutical administration. The use of such medium and agents for pharmaceutically active substances is well known in the art. Except for when any medium or agent is compatible with the VSV vector, such media are used in the immunogenic compositions of the invention. The supplementally active compounds can also be incorporated into the compositions. Thus, an immunogenic composition of the VSV of the invention is formulated to be compatible with its proposed route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal) and mucosal (e.g., oral, rectal, intranasal, buccal, vaginal, respiratory).

The suspensions or solutions used for parenteral, intradermal or subcutaneous application, include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; pH regulators such as acetates, citrates, or phosphates and tonicity adjusting agents such as sodium chloride or dextrose. The pH is adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where they are soluble in water), or d sterile dispersions and powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ™ (BASF, Parisppany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be saline and must be fluid in the extent that there is easy syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol and the like), and suitable mixtures thereof. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms is achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions is carried approximately including in the composition, an agent which retards absorption, for example, aluminum monostearate and gelatin. Syringe injectable solutions are prepared by incorporating the VSV vector in the required amount (or dose) into an appropriate solvent, with one or a combination of ingredients listed above, as required, followed by filtered sterilization. In general, the dispersions are prepared by incorporating the active compound in a sterile vehicle, which contains a basic dispersion medium and the other ingredients required for those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, which provides a powder of the active ingredient plus any of the desired additional ingredients of a previously sterile filtered solution thereof.

For administration by inhalation, the compounds are supplied in the form of a pressurized container aerosol spray or dispenser, which contains a suitable propellant (eg, a gas such as carbon dioxide or a nebulizer). Systemic administration can also be by mucosal or transdermal means. For mucosal or transdermal administration, the appropriate penetrants to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for mucosal administration, detergents, bile salts and fusidic acid derivatives. Mucosal administration is done through the use of nasal sprays or suppositories. The compounds are also prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides), or retention enemas for rectal delivery. In certain embodiments, it is advantageous to formulate oral or parenteral compositions in the form of a dosage unit for ease of administration and uniformity of dosage. The dosage unit form as used below, refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the technique of compounding, such as a Active compound for the treatment of individuals. All patents and publications cited in this document are hereby incorporated by reference.

D. EXAMPLES The following examples are carried out using standard techniques, which are well known and routine by those skilled in the art, except where otherwise described in detail. The following examples are presented for illustrative purposes, and should not be constructed in any way limiting the scope of this invention.

EXAMPLE 1 Materials and methods Cytoplasmic Extremity Mutants of VSV Protein G The methods used for the generation of cytoplasmic limb mutants of the G protein of the present invention are known in the art and are described in detail by Schnell et al., (1998). These G protein mutants retain either a single amino acid (G (ct-1)) or nine amino acids (G (ct-9)) in the cytoplasmic G domain, compared to the twenty-nine amino acids in the cytoplasmic limb domain of the Indiana strain of native VSV (SEQ ID NO: 1). Truncations of cytoplasmic limb were generated by moving the translation stop codon either 60 nucleotides or 84 nucleotides (ie, nine amino acids of cytoplasmic limb and one amino acid of cytoplasmic limb, respectively), upstream of the authentic arrest codon and resulting in the truncation of protein G.

Mixed Mutants of N Gene of VSV Gene translocation mutants of the G gene (mixtures of N) were generated by repositioning the N gene as either the second, third or fourth gene from the 3 'end of the virus genome. For example, the order of the authentic gene for the 3'-NPMGL-d 'of the native VSV was mutated to 3'-PNMGL-5' and 3'-PMNGL-5 '. The translocation of the additional N gene separated from the unique 3 'RNA transcription promoter causes a proportionate fall in the level of N gene expression (eg, see U.S. Patent No. 6,696,629, specifically incorporated herein by reference in its entirety). A reduction in the level of N protein in infected cells, decreases viral nucleocapsid formation, finally reducing the ratio of genome replication and formation of virus particles. The methods used for translocations of the N gene are described below. For the first stage in the production of the N gene changes, the N gene was completely removed from the cDNA of the full-length virus genome, with the result that the P gene was then immediately adjacent to the leader virus, instead of the gene N. To suppress the N gene, two PCR products were made with a full-length genome cDNA as a template. The first PCR product contains sequence stretching of a native BsaAl site upstream of the T7 promoter, to the terminus of the virus leader and a downstream BsmBI site added. The second PCR product contains sequence stretching of the natural Xbal site in the P gene to the start signal of transcription to the P gene, adjacent to an upstream BsmBI site added. The BsmBI sites are arranged in a way that both PCR products can be joined without seam joints (after digestion and ligation), to give a DNA fragment containing the virus leader immediately adjacent to the P gene. DNA is then ligated into the Xbal / BsaAl sites of the full-length genome cDNA, effectively removing the n gene from the virus genome. In the next step of generation of N gene changes, the N gene is inserted between either the P and M genes, or the M and G genes, or the G and L genes of the deleted genome cDNA. For insertion of the N gene between the P and M genes, three PCR products were prepared with the full-length genome cDNA as a template. The first PCR product contains sequence stretching of the natural Xbal site in the P gene, to the start signal of the transcription of the M gene, with. a BsmBI site with aggregate flanking. The second PCR product contains sequence stretching of the start signal from the transcription of the N gene to the conserved TATG sequence, adjacent to the polyadenylation signal -AAAAAAA- in the N gene, with an aggregated flanking BsmBI site. The third PCR product contains the stretching of the natural Mlul site sequence at the beginning of the G gene to the conserved polyadenylation signal TATGAAAAAAA of the P gene, with an aggregated flanking BsmBI site. Three fragments are then digested with BsmBI, and religated to form a single DNA fragment with the N gene flanked by part of the P gene and the M gene. This DNA fragment is then deferred with Xbal and Mlul and ligated into the Xbal / sites. MIul of the delta-N virus genome to form the 3'-PNMGL-d 'cDNA. To generate the 3'-PMGL-5 'genome cDNA, two separate PCR products were prepared. The first PCR product contains sequence stretching of the natural Xbal site in the P gene to the transcription initiation signal (d'AACAG-3 ') of the G gene, with an aggregated flanking BsmBI site. The second PCR product contains the complete N gene sequence of the transcription initiation signal, with a BsmBI site upstream of flanking, to the stop signal / polyadenylation of the N gene transcript, with stretch of the sequence of flanking the start signal of G transcription to the natural Mlul site in the G gene. The G gene specific sequence was added to the N gene sequence as part of one of the PCR primers. Both PCR products were digested with BsmBI and ligated to form a single DNA fragment, which was then digested with Xbal and Mlul and ligated into the Xbal / MIul sites of the deleted N genome cDNA to give an array of the d 'gene -PMNGL-3 '. To generate a cDNA of the genome d'PMGNL-5 ', three PCR products were prepared from a cDNA template of the complete genome. The first PCR product contains a sequence stretch from the natural Swal site in the G gene to the transcription start signal for the L gene, flanked by an aggregated BsmBI site. The second PCR product contains the sequence for the complete N gene, from the transcription start signal to the transcription stop signal, flanked at both ends by aggregated BsmBI sites. The third PCR product contains a sequence stretch of the transcription start of the L gene, flanked by an aggregated BsmBI site, to a natural Hpal site in the L gene. All three PCR products were digested with BsmBI and ligated to form a single DNA fragment, which was then digested with Swal and Hpal, and ligated into Swal / Hpal sites of the deleted N-genome cDNA, resulting in an array of the 5'-PMGNL-3 'gene. In all three genomes rearranged to the sequence integrity of each gene and flanking regulatory sequences are identical to the unaltered virus; only the position of the N gene is different.

Combination of Cytoplasmic Extremity Mutations of Protein G and Mixed Mutations N The combination of both the N-mixtures and the cytoplasmic limb truncations of the G protein result in dually mutated genomes (ie, two kinds of mutation), for example 3'-PNMG (ct-9) L-5 \ 3'-PNMG (ct-i) L-5 'and 3'-PMNG (ct-9) L-d', 3'-PMNG (ct-1) L-d '. Double mutant genome cDNAs were constructed by changing the natural G gene in the mixed N genomes, with either the truncated G (Cn) or G (Ct-g) d genes described above. The change was made by digestion of donor cDNAs (d'-NPMG (ct-i) L-3 'and d'-NPMG (Ct-9) L-3') with Mlul and Hpal, followed by ligation of the G genes. truncated, purified in the Mlul / Hpal sites of the mixed cDNA genomes N, triple purified plate, amplified and characterized in cell culture by plate size and growth kinetics 0 as described below.

Mutations of the non-cytopathic M gene The M gene of the VSV encodes the protein matrix (M) of the virus, and two small structure polypeptides (M2 and M3). The M2 and M3 polypeptides were transferred from the same open reading structure (ORF) as the M protein, and lack the first amino acids 33 and 51 respectively. A recombinant VSV vector comprising non-cytopathic M gene mutations (ie, VSV vectors that also do not express M2 and M3 proteins) was generated as described below, and further comprises one or more additional mutations, thereby resulting in a VSV vector that is highly attenuated in cell culture and in animals. The non-cytopathic M gene mutations (M (ncp)) were generated, which result in the conversion of methionines 33 and 51 to alanines (M33A, M61A) using a PCR-based cloning strategy, where the nucleotide substitutions required (AUG to GCT) were incorporated into the PCR primers (Jayakar and Whitt, 2002; Jayakar et al., 2000). The resulting PCR products containing the M33, d1A mutation were then cloned into the full length VSV cDNA genome, allowing rescue of virus not expressing the M2 and M3 polypeptides. The M33, d1A mutations present in the cDNA of the recombinant VSV vector designated by Jayakar and Whitt, were transferred to cDNA of the VSV vectors by exchanging the Xbal-Mlul fragment (which runs through the entire M gene and part of the P gene). The cDNA fragment does not change the result in any additional amino acid encoding changes on and before the M33,51A mutations.

Combination of Protein G Tip Mutations and Non-Cytopathic M Gene Mutations The combination of both G protein cytoplasmic limb truncations and non-cytopathic M gene mutations results in doubly mutated genomes (ie, two kinds of mutations), by example 3'-NPMncpGct-1 L-5 'or 3'-NPMncpGct-9-L-d'. The double mutant genome cDNAs were constructed by changing the cDNA of the M gene containing the mutations that give rise to the non-cytopathic phenotype, into full length genome cDNAs containing either G (Ct-i) or G (Ct-9) mutations ). In each case, the stretching of the changed cDNA fragment from the unique Xba I site in the P gene to the unique Mlu I site in the untranslated region d 'of the G gene, includes the complete non-cytopathic M gene sequence. As previously described, the non-cytopathic M protein differs from the M protein, replaced, by only two amino acid substitutions (M33A and M61A), which give rise to the non-cytopathic phenotype. These doubly mutated genomes are then further modified by insertion of the gag HIV-1 gene at position 5 in the genome, between the G and L genes, to allow the expression of the gag protein for immunogenicity studies. As for other virus rVSV vectors, the gag gene was cloned into the unique Xho I Nhe I sites at position 5 of the genome cDNA.

Temperature Sensitive Mutations of the VSV N Gene and / or Temperature Sensitive Mutations of the VSV Gene L A Gag HIV protein encoding the recombinant VSV (rVSV) of the first 3 'cistron in the viral genome (rVSV-Gagí) was modified by replacing in N gene and / or L gene with sequences encoding homologs derived from mutants (ts) known to be temperature sensitive from the biologically derived VSV (Pringle, 1970). The resulting vectors, (i) rVSV-Gag-itsN (ie 3'-gag? -N (tS) PMGL-5 ') containing the N ts gene of the ts41 strain of the VSV, (ii) rVSV-Gag -itsL (ie, 3'-gag? -NPMGL (tS) -d ') containing the L gene of the ts11 strain of the VSV and (iii) rVSV-Gag ^ sN + L (ie, 3'-gag) ? -N (ts) PMGL (ts) -d ') containing both the N ts gene of the ts41 strain of the VSV and the L gene of the ts11 strain of the VSV. Strains ts41 and ts 11 of the VSV are also known in the art as tsG41 and tsG11, respectively. Both strains of the biologically derived ts gene donor were isolated by Pringle (Pringle, 1970) then subjected to a VSV adapted to the laboratory (the Glasgow strain of the Indiana Serotype) to chemical mutagenesis, Pringle also mapping the ts mutations to the N gene or L. Genes ts41 N and ts 11 were strained from infected cell RNA. Briefly, BHK cells were infected with ts11 or ts14 at permissive temperature (31-32 ° C). The infection was also allowed to proceed until the cytopathic effect was evident in more than 7d% of the cell monolayer, at which time the total RNA was extracted and purified. The RNA was then reverse transcribed using gene-specific primers for direct cDNA synthesis after which the cDNA was amplified by PCR. The amplified cDNAs were then cloned into the genomic cDNA of the rVSV vector and verified by sequence analysis. The complete genomic sequence of ts11, ts41 and its progenitor strain (Glasgow) was determined by identifying coding changes that contribute to the ts phenotype. By comparing coding sequences of the vector structure of rVSV, ts Pringle mutants, and Glasgow progenitor virus, it is possible to predict such coding changes that contribute to the ts phenotypes of the vectors rVSV-GagitsN, rVSV-GagitsL and rVSV-Gag ^ sN + L. Table 3 is a comparison of amino acid sequences of the N protein. It is apparent from the data that replacement of the N gene of the rVSV vector with the ts41 homologue resulted in the replacement of amino acid 4. Either of these changes it can affect the function of the N protein in the context of the genetic structure of the vector and contributes to the ts phenotype. It is notable that only one change is critical (Tyr to Cys at position 74, shows residuals in italics) that distinguishes ts41 from its progenitor virus (Glasgow), suggests that this substitution can be a determinant ts.

TABLE 3 Comparison of n VSV proteins Similarly, Table 4 provides the comparison of the L protein. Replacement of the L gene in the rVSV vector with the ts11 complement results in changes encoding 13 amino acids. As mentioned above for the N gene, any of these coding changes may contribute to the observed ts phenotype produced by the replacement of the L gene, but several of these coding mutations (shown in italics) are of greater interest because they also differentiate ts11 from its Glasgow progenitor virus, potentially identifying these amino acid substitutions as key contributors to the ts phenotype.

TABLE 4 Comparison of VSV protein 1 G / Gen In certain embodiments, a genetically modified VSV of the invention comprises a mutation in the G gene, ein the encoded G protein has a mutation in the backbone region near the membrane of the G protein ectodomain, referred to as G protein The truncal G mutation is introduced by replacing the G gene in the genetic structure of the VSV vector XV (Schnell et al., 1996) with a modified G gene encoding the G trunk. The G trunk (Robinson, 2000) is composed of 108 to 512 amino acids of the G protein including: 1) the first 17 amino acids of the G protein, which span the target signal sequence of the polypeptide for membrane insertion; 2) 42 amino acids of the extracellular domain near the membrane referred to as the trunk; 3) amino acid 20 of the domain encompassing the membrane; and 4) the amino acid 29 of the carboxy terminal intracellular extremity. This configuration of the core G polypeptide contains sufficient G protein sequence for mediated maturation of viral particles, but lacks the sequences necessary to act as a cell binding protein. Consequently, cells infected with a G trunk vector will express viral proteins and the encoded foreign antigen, but will produce viral particles of offspring that are non-infectious because the G backbone vector does not encode a fully functional G protein. To produce particles of the truncated G vector containing the functional G protein, necessary to infect a target cell, a full-length G protein in trans must be provided. This can be achieved during virus rescue and subsequent vaccine production by one of several procedures: 1) cell lines expressing the G protein can be developed; 2) a complemented viral vector expressing the G protein can be employed, such as adenovirus, MVA or VEE; or 3) cells used for production can be transfected with a plasmid DNA vector or mRNA encoding the G protein. Currently, the truncated G vector is produced by temporary complementation in cells transfected with a plasmid designated to express the G protein. This avoids the need to generate cell lines that express the G protein, which are difficult to produce because the G protein is toxic, and also avoids the introduction of a biological reagent similar to the herpes virus in the production procedures. In some configurations of the G trunk vector, the cistrons encoding the viral proteins have been mixed upstream to allow the insertion of a foreign gene into the first position of the genome. This attenuates the virus and places the foreign antigen gene near the promoter ensuring high levels of expression. As described above in Section A1, the insertion of the HIV gag gene (or any other gene) into the VSV genome at position 1 (3'-gagrNPMGL-5 '), results in a mutation of the gene mix , e the genes N, P, M, G and L are each moved from their native type positions to more distal positions in the genome. Thus, the combination of both the G (troncai) mutation and the gag insertion in the VSV genome at position 1 (gag-i) results in a double-mutated 3'-gagr NPMG (troncai) L-5 'genome. .

Rescue of the Vesicular Stomatitis Virus in 293 Cells Successful rescue of the VSV of 293 cells was achieved using the heat shock system / plasmid-17 described in the international application WO 2004/113517 (specifically incorporated herein by reference) , in accordance with the protocol reviewed below.

Materials DNA Plasmids: 1) full-length viral genomic cDNA, 2) pT7-N, 3) pT7-P, 4) pT7-L, d) pT7-M, 6) pT7-G and 7) pCI-Neo-bclT7 (p0061).

Calcium-carbonate transfection reagents: 1) BES 2X buffered saline: BES 60mM (pH 6.96-6.98), 280 mN NaCl, 1.5 mM Na2HOP4, 2) 2.6 M CaCl2 and 3) buffered saline washer solution Hepes (HBS): 20 mM hepes (pH 7.0-7.5), 140 mM KCl, 1 mM MgCl2.

Cell Culture Solutions: 1) DMEM supplemented with 10% heat-inactivated FBS, certified (DMEM / FBS), 2) Iscoves Modified Minimum Essential Medium (IMEM) supplemented with 10% heat-inactivated FBS, certified (IMEM / FBS) ), 3) Poly-L-Lysine: 0.01% in H20, 4) PBS and 5) Porcine trypsin / EDTA.

PROCEDURES 293 Cell Culture: 293 cells can be difficult to grow, and there are a number of different methods to manipulate them. The current method has been successfully used as part of a rescue for the vector constructs of the VSV and modified VSV.

Routine subculture: 1) The medium was removed and the confluent monolayer washed (10 cm of plate) with d ml of hot PBS; It was pipetted gently along the side of the disc to prevent separation of the cells (293 cells were left at room temperature for a prolonged time, or detached in medium that became basic (red)). 2) 2 ml of trypsin was added gently and the plate was tilted to cover the complete monolayer. The trypsin was aspirated and plate 6 was allowed to remain at room temperature for about one minute. The plate was tilted at an angle of 46 degrees and capped against the working surface of the cover to detach the cells. If the cells do not detach, incubate another minute at room temperature (ensuring that the cells detach at this stage so that vigorous pipetting is avoided). 3) D ml of DMEM / FBS was gently added and pipetted up and down to disperse the cells. 4) 1 ml of cells was added to a plate containing 9 ml of DMEM / FBS. d) It was incubated at 37 ° C, 5% C02.

Subculture for transfection: 1) The desired number of plates was coated with poly-L lysine. About 3-4 ml of 0.001% poly-L lysine was added per plate and allowed to stand at room temperature for at least 30 minutes. The poly-L lysine solution was aspirated. The plate was rinsed with d ml of the medium. 2) The cells were trypsinized as described above. A separation ratio was used that provided a 50-0 75% confluent plate the next day (1: 3 to 1: 6). 3) After separating the cells, IMEM / FBS was added and the cells were transferred to the coated plate containing 9 ml of IMEM / FBS. It seems important to detach the cells and allow growth overnight in IMEM / FBS before transfection. d 4) Incubation at 37 ° C, C02 at 5%.

Transfection: 1) 1-3 hours before transfection; The cells were fed with 9 ml of IMEM / FBS and the cells were incubated in an incubator at 32 ° C set at I 3% C02. 2) The calcium-phosphate-DNA transfection mixture was prepared as follows: a) The following DNAs were combined in a 5 ml polypropylene tube: (i) 8 μg T7-N; (ii) 4 μg T7-P, (ii) 1.2 μg T7 L, (iv) 1.0 μg T7-M, (v) 1.0 μg T7-G (vi) 10 μg of the viral genomic cDNA clone and (vii) 10 μg of the expression vector hCMV-T7. b) The volume was adjusted to a final volume of 460 μl with 5 water. c) 50 μl of 2.5 M CaCl2 was added. d) While the tube was gently vortexed, 500 μl of 2XBBS was added after the tube was left at room temperature for 16-20 minutes. 0 3) The cells were removed from the incubator and the calcium-phosphate-DNA mixture was slowly added to the culture medium and gently stirred to distribute the precipitate. Immediately the cells were returned to the incubator at 32 ° C-3% C02. 4) Three hours after starting transfection, the disks of the culture were sealed in a plastic bag and completely submerged in a water bath set at 43 ° C for 2 hours to induce the cell heat shock response. d) After the heat stroke, the cells were returned to the incubator at 32 ° C / 3% C02 and the incubation continued overnight. 6) The next day, the cells were washed 2 times with HBS and the cells were fed with 10 ml of IMEM / FBS. They were incubated at 37 ° C, in 5% C02. 7) At 48-72 hours after the start of transfection, sufficient T160 flasks containing 20 ml of DMEM / FBS were established for transfer of transfected cells to the larger vessel. A T150 flask was transfected for each 10 cm plate. 8) Transfected 293 cells were transferred by gently pipetting the culture medium over the monolayer to entrain it from the cell surface. Avoid vigorous pipetting and use only enough force to drag the cells. After the cells are discharged, pipette up and down approximately d times, to reduce the size of the cell clumps, then transfer the medium and cells 0 to a T160 flask containing the 20 ml of IMEM / FBS. 9) After 6 hours, replace the medium with fresh DMEM supplemented with 10% FBS (note that this stage can be delayed up to 24 hours if the cells do not adhere to the plate.) This stage has also been successfully skipped. . 5 10) Monitor the cells for 5-7 days to detect evidence of cytopathic effect. 11) When the CPE seems evident, transfer 50 ul of supernatant medium to the cavity in a six-well plate containing medium and a monolayer of established Vero cells. The CPE must be visible the next day if the rescue has occurred. (Note that this stage is important because the 293 cells at times break off from the surface of the T150 flask and appear to infect the VSV when they are not currently). 12) After transferring the small sample to the Vero cell monolayer, collect the cells and the medium from the T150 flask and freeze at -70. The 293 cells can generally be collected by pipetting the medium onto the monolayer to detach the cells.

Rescue of Vesicular Stomatitis Virus in Vero Cells Solutions The following solutions were generated useful for the transfection of host cells: 1) Solution A 2XBBS (for I) (saline buffered with 2XBES) of 280 mM of NaCl [16.4 g, NaCl (or 56 ml of 5 M NaCI)], 50 mM of BES [10.7 g of Bes (free acid form)], and 1.5 mM of sodium phosphate [0.21 g of Na2HP0]. The BBS solution was adjusted to pH 6.95-6.98 with NaOH. The solution is then sterilized filtered and frozen stored. 2) A CaCl2 2.5M solution of 36.8 g per 100 ml of total volume was prepared and stored at -20 ° C. The solution is sterilized filtered using nitrocellulose. Cellulose acetate filters are avoided because they coagulate. Alternatively, the transfection solutions are autoclaved for sterilization. However, the latter procedure may be less desirable, because the 2XBBS solution may change slightly during autoclaving. The following solutions are generally useful for the medium: 1) A DMEM + FBS solution of DMEM (high glucose with glutamine, Gibco / BRL, [Grand Island, NR]), supplemented with 10% heat-inactivated FBS and certified, and 10-20 μg / ml (optionally up to 60 μg / ml) of gentamicin. 2) A MEM + FBS solution of MEM (supplemented with glutamine, non-essential amino acids, FBS inactivated by 10% heat and certified, and 10-20 μg / ml (optionally up to 60 μg / ml) pH regulator Hepes 20- 25 mM; Gibco / BRL) (Grand Island, NY), and which optionally includes Fungizona 1X). 3) An HBS solution of pH-Hepes buffer saline solution, 20 mM hepes, pH 7.0, 150 mM NaCl, 1 mM MgCl 2.

Methods A generally useful host cell can be selected from divided Vero cells, which are placed in DMEM + FBS the day before transfection, so that they will be approximately 50% confluent [80-90% for RSV], the next day (in plates of six cavities or flasks of 12.5 cm2). Higher cell densities work less effectively. The next day, each culture is fed 1-4 hours before transfection with 4.6 ml of DMEM + FBS. The cells are then transferred to a C02 incubator set at 3% C02 and 32 ° C. Ver cells can be grown for a prolonged period at night, as soon as they are approximately 50% confluent at the time of transfection.

A precipitate of CaCl2 / phosphate is obtained as follows: BBS and CaCl2 are maintained at room temperature before initiation. The mixture of DNA is prepared in a polypropylene tube of d ml containing a total volume of 260 μl, with the plasmid DNA 2-20 μg total and 2d μl of Ca2Cl2. Full length rescue DNAs include, d μg of a full-length cDNA construct for N protein of 400 ng, protein P of 300 ng, protein L of 100-200 ng, and plasmid pCI-Neo-Bcl- T7 of 6-10 μg of VSV (SEQ ID NO: 1, Figure 2). The efficiency of the rescue in Vero cells is low, so 3-6 cavities are transfected per full-length construct to be rescued. After all the DNA / CaCl2 solutions are prepared, 2XBBS is added. This is usually done by gently shaking a tube by vortexing continuously at low speed and adding 250 μl of 2XBBS per drip down the side of the tube. This is repeated for all tubes, which are allowed to stand at room temperature for an additional 15-20 minutes, to allow DNA-Calcium-Phosphate to precipitate to form. After incubation at room temperature, the precipitate is added dropwise to the cell culture medium and distributed evenly by tilting the plate. The medium is then incubated for three hours in an incubator set at 3% C02. A level of C02 at 3% is important for the transfection technique of BBS / CaCi2; The C02 at 5% works very little, if at all. C02 at 3% controls the pH of the medium and allows the formation of a precipitate of calcium-phosphate-DNA in the medium.

A thermal shock process is then optionally carried out, for example, at three hours after the initiation of transfection. The cells are transferred to a water bath set at 44 ° C. The cells are sealed in a plastic storage bag, so that the cultures can be completely immersed in water. After three hours at 44 ° C, the cells are transferred again in an incubator at 32 ° C set at 3% C02 and the incubation is continued overnight. The next day, the transfection medium is removed and the cells are washed twice with HBS. After washing, 2 ml of DMEM + fresh FBS are added. The PBS and Hank pH regulators operate poorly for the washing step, probably because the phosphate in these pH regulators causes more CaCl2 to precipitate from the transfection medium. A co-culture procedure is then optionally performed. Transfected cells are harvested at 48-72 hours after transfection, scraping them in the medium and transferring the most medium cells to a T25 flask containing a monolayer at 60% confluence of Vero cells. Six hours after starting this co-culture, the medium is replaced with 4 ml of MEM + FBS. The cultures are then incubated for five days. If the medium begins to appear consumed during this incubation period, 2 ml of the medium are removed and replaced with fresh MEM + FBS. It is not recommended that the entire medium be replaced, to conserve any lesser amount of virus that is generated during the rescue, which may be in the middle. During this co-culture phase, the CPE may become evident, but this is usually not the case. If CEP is not evident, the rescue can be continued. The cells are collected five days after starting co-culture. First, add O.d ml of 2.18 M sucrose, 37.6 mM KH2P04, 71.0 mM K2HP0, 49.0 mM sodium glutamate to the medium and mix by swirling the flask. The cells are then scraped into the medium, pipetted up and down to mix, and then aliquoted into freezing tubes for shipping and then rapidly frozen in a dry ice / ethanol bath and stored at -80 ° C.

Purification of the VSV Vector The rescued VSV vectors are plaque purified from the supernatants of the transfected cells. After three successive rounds of plate purification, the virus was amplified in BHK cells to produce a seeded base solution, which in turn was further amplified in BHK cells to produce a working base solution virus. To prepare large quantities of virus for animal experiments, the working base solution was used to infect 10-20 T-150 flasks of confluent BHK cells, at a multiplicity of infection (MOI) of 0.5-1.0 plaque forming units (pfu) /cell. After 48 hours at 32 ° C, the supernatants from infected cells were clarified by centrifugation at 4,000 x g. The viruses were then concentrated from the supernatants by centrifugation in a SW 28 rotor at 25,000 rpm for one hour, through a 10% sucrose cushion. The pellets of the virus were again suspended in phosphate pH-regulating salt (PBS) and snap-frozen in a dry ice / ethanol bath. The concentrated virus base solution was then titrated in Vero cell monolayers to determine the number of infectious particles in the preparation.

Virus titration The number of infectious particles of the virus in a virus preparation was determined by a standard plaque assay. Briefly, Vero cell monolayers were infected at night, recently confluent in six-well plates, infected with 10 parts of serial dilutions of the virus preparation. To do this, the growth medium was aspirated from the cell monolayers and 100 μl aliquots of each dilution of virus in DMEM was transferred in triplicate to the center of the cell monolayers. To prevent cellular desiccation, 400 μl of DMEM were then added to each cell monolayer and the plates were kept at room temperature for fifteen minutes, followed by a thirty minute incubation at 37 ° C, 6% C02, with occasional tilting. The virus inoculum was then removed and each cell monolayer was overlaid with 3 ml of 0.8% agarose in DMEM. The plates were then incubated at 37 ° C, C02 at d% for 1-4 days to allow plaque formation. The agarose plugs were then removed, and the cells were stained with crystal violet (2% crystal violet in 50% methanol), for ten minutes at room temperature. The excess dyeing was then removed and the cell monolayers were rinsed uniformly with water. The virus plates were then visualized in the cell monolayer as small holes that do not stain blue.

Quantification of viral RNA by Real Time PCR A quantitative real-time PCR assay (RT / PCR) was used for the detection and quantification of VSV genomes in the tissue of animals. The assay uses a 2-step RT / PCR procedure, which specifically detects the genomic RNA of the virus in the negative sense and uses a synthetic oligonucleotide of the complete amplicon for the development of a standard curve. Briefly, the brain tissues of monkeys, ferrets and mice were homogenized as a 20% suspension in P / V in SPG. The suspension was centrifuged at 3,000 x g for fifteen minutes to pelletized particulate material. The supernatant was then further centrifuged at 14,000 x g, and the total RNA was extracted from the resulting supernatant. This RNA was used as a template for reverse transcription, with the virus-specific primers and the products were then used for the real-time PCR assay.

Determination of 60% lethal dose (LDdO) of VSV vectors in mice The mouse LD50 model was used as a measure of the relative attenuation of the VSV vectors. Several log-fold dilutions of the S'-PMGíct-DL-d ', 3'-PNMG (ct-i) L-5' and 3'-PMNG (ct-1) L-5 \ of the native VSV , were injected intracranially in Swiss Webster female mice of four and one and a half weeks of age (6-10 mice per group). The mice were left for weight loss, paralysis and death (LD50) for three weeks. The LD50 was calculated from the cumulative percentage of mortality by the method of Reed and Muench.

Mouse Immunogenicity Studies Mice (n = 15) were immunized intramuscularly with 1x107 pfu of the indicated VSV vectors (Indiana serotype) set forth in Example 4. Splenocytes from a series of mice ("Primer" n = 5) were isolated at the peak of the effector phase 7 days after the start. Two sets of mice (n = 10) were boosted with 1x10 7 pfu of the indicated VSV vector (NJ-G-switched version). Splenocytes from a series of mice ("Reinforcement", n = 5), were isolated at the peak of the effector phase 5 days after reinforcement. Splenocytes from another series of mice (Memory, n = 5), were isolated during the memory phase 30 days after reinforcement. The frequencies of Gag specific CD8 T cells were determined by tetramer staining. The secretion of IFN-α was determined Specific Gag by ELISPOT after stimulation overnight with the immunodominant peptide gag.

EXAMPLE 2 Characterization of VSV mutants Substantial differences were observed between the plate sizes of the two VSV vectors of the combined mutation class described in Example 1 (ct truncation of the G protein / N mix) against the VSV vectors of the single class mutation ( d). Typically, the VSV vectors of the single-class mutation, formed countable size plates in a twenty-four hour plate assay, while some of the G protein / N-truncated protein truncation vectors require three to four days to form plates of equivalent size. The relative differences in plate size for the VSV vectors are also parallel relative differences observed during the cell culture growth kinetics studies (Figure 1, to Figure 3).

TABLE 5 Relative size of plate of the VSV vectors EXAMPLE 3 Studies of neurovirulence of VSV The synergistic attenuation of VSV comprising a combination of two or more kinds of mutation, relative to the mutant vectors of the single-class VSV, was evaluated in a series of mouse neurovirulence studies; ferrets and monkeys, the methods which are described in Example 1. Mice are highly permissible for VSV replication and this property allows them to be used to discriminate different levels of virus growth and attenuation. A different pathogenicity / attenuation gradient was observed in mice for the different VSV vectors (Table 6 and Table 7). For example, LD50 in mice inoculated intracranially with 3'-NPMGL-5 ', 3'-NPMG (ct-i) L-5', 3'-PNMG (ct-1) L-5 'or 3'-PMNG (ct-i) L-5 '(Table 6), indicate the following relative attenuation gradient: 3'-PMNG. { ct-1) L-5 '(LD50 = 2 x 105) > 3'-PNMG (ct-i) L-d '(LD50 = 1 x 104) > 3'-NPMG ^ -DL-d * (LD50 = 14.5) > 3'-NPMGL-5 '(LD50 = 3.2).

TABLE 6 Number of killed or paralyzed mice 6 mice were inoculated intracranially (IC) with any of the above vectors.

LD50 in mice injected intracranially with VSV vectors having zero (native VSV), one, two, three and four (gag gene insertion) mutation classes, shown below in Table 7, also exhibited a gradient of attenuation Similary. In addition, mice injected intracranially with vectors 3'-gag? -PMNG (Ct-i) L-5 ', 3'-gagrN (ts) PMGL (tS) -5, S'-gagrNPMGL ^ -d', a'- gag NPMGfocp ctDL-d, 3'-gag PMNG (ct9) L (str5 'and 3'-gag? -NPMG (trQncai) L-5' of the VSV, did not present mortality.

TABLE 7 Intracranial neurovirulence of VSV vectors in mice The histopathological data of Cinomolgus monkeys inoculated intratalmically with the same series of vectors indicates a very similar attenuation gradient. Both series of animal data were further corroborated by the results of a series of ferret neurovirulence studies, in which the infectious virus and genomic RNA levels present in the brains of intracranially inoculated animals were periodically measured by the plaque assay and Real-time PCR, respectively. Collectively, these data demonstrate that the combination of two or more mutation classes has an attenuation level that is substantially greater than the VSV vectors of the single mutation class. The titers of mouse LD50 strongly indicate that there is a potent synergistic effect in the attenuation by combining two different classes of mutation in the same VSV vector.

EXAMPLE 4 Enhanced immunity of attenuated VSV vectors The immunogenicity of attenuated VSV vectors 3'-gag -? - were compared with the prototype vectors of VSV 3'-NPMG-gag5-L-5 'and 3'-NPMGL-5'. Mice were immunized with one of the above VSV vectors, as described in Example 1. Attenuated VSV vectors induce immune responses that were stronger than those induced by the prototype VSV-Gag5 vector (3'-NPMG -gag5-L-5 '). The most notable was the 3'-gag? -PMNG. { ct9) L-d ', which statistically induces Gag-specific T-cell frequencies significantly higher than those induced by the prototype when assessed after priming and reinforcement, as well as, during the memory phase of the answers (Table 8).

TABLE 8 Frequencies of G8-specific CD8 T cells = Significantly higher response than that observed for 3'-NPMG-gag5-Ld '(Student's t test, p <0.05). 3'-gag? -PMNG (Ctg) L-5 ', also induces secretion of IFN-? provided higher than that induced by the prototype 3'-NPMG-gag5-L-d '(Table 9). The responses to 3'-gag NPM (ncp) G (Cti) L-5 'and 3'-gag N (tS) -PMGL (ts) -d', are also provided higher than that induced by the prototype 3'- NPMG-gag5-L-5 '(Table 9). 96 TABLE 9 ELISPOT of IFN-? of GAG * = significantly higher response than that observed for 3'-NPMG-gag5-L5 '(Student's t test, p <0.05).

EXAMPLE 5 Immunogenicity of intramuscular and intranasal delivery of attenuated VSV vectors expressing HIV GAG in rhesus macaques The following studies were designed to measure the immune responses elicited in rhesus macaques, after immunization with attenuated VSV vectors expressing HIV gag protein. The study set forth in Table 10 was made with a total of twenty-four genetically unselected male rhesus macaques, where each group of animals (ie, Groups 1-6) is immunized either intramuscularly or intranasally with one of the following VSV vectors at a dose of 1 x 107 (pfu): (TsN + L), 3'-gagr PMNG (ct9) L-5 '(N4CT9) or 3'-NPMG-gag5-L5' (Gagd). The study set forth in Table 11 is performed with a total of twenty-four genetically unselected male rhesus macaques, wherein each group of animals (ie, Groups 1-8) is immunized either intramuscularly or intranasally with one of the following vectors. of the VSV at a dose of 1 x 107 (pfu): 3'-gagrNPM (ncP) G (Cti) Ld '(MncpCTI), 3'-gag NPMGdroncaDL-d' (Trunk G), S'-gag PMNG ^ DL -d '(N4CT1) or 3'-NPMG-gag5-L-5' (Gagd). In general, the following tests were used to examine the systemic and humoral immune responses and imparted by the VSV of each animal: Cellular Immune Responses: ELISPOT responses of INF-? Specificity of the HIV gag peptide and IFN-? ELISPOT responses? specific peptide N of the VSV. Humoral immune responses: gag antibody titers of serum anti-HIV by ELISA, serum anti-VSV antibody titrators by serum anti-VSV neutralization antibody titers and ELISA.

TABLE 10 Primate immunogenicity study The IM route is intramuscular The IN route is intranasal TABLE 11 Primate immunogenicity study The IM route is intramuscular The IN route is intranasal References U.S. Patent 5,268,464 U.S. Patent 5,666,747. U.S. Patent 5,789,166 U.S. Patent 6,391, 548 U.S. Patent 5,817,879 U.S. Patent 6,168,943 U.S. Patent 6,596,529 U.S. Patent 6,033,886 U.S. Patent 6,673,572 U.S. Provisional U.S. Patent 60 / 477,389 Ahmed and Lyles, "Identification of a Consensus Mutation in M Protein of Vesicular Stomatitis Virus from Persistently Infected Cells that Affects Inhibition of Host-directed Gene Expression "Virology, 237 (1): 378-88,1997. Atkinson eí a /., United States. Annu. Rev. Med., 43: 451-463, 1992. Ball eí a /., "Phenotypic consequences of rearranging the P, M, and G genes of vesicular stomatitis virus" Journal of virology, 73 (6): 4705-4712, 1999. Bellini et al., Emerging Infectious Diseases, 4: 29-35, 1998. Canto et al., "Status Spongiosus Resulting from Intracerebral Infection of Mice with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus", Br. J. exp. Path., 57: 321-330, 1976. Collins et al., Adv. Virus. Res., 54: 423-451, 1999. Collins eí al, Respiratory Syncytial Virus. In "Field's Virology", 3rd Edition, Lippincott-Raven, 1443-1475, 2001. Cutts et al., J. Infectious. Dis., 170: S32-S41, 1994. Evans et al., "Temperature-sensitive mutants of complementation group E of vesicular stomatitis virus New Jersey serotype possess altered NS polypeptides", Journal of Virology, 31 (2): 325- 333.1979. Finke and Conzelmann, "Ambisense gene expression from recombinant rabies virus: random packaging of positive- and negative-strand ribonucleoprotein complexes nto rabies virions", J. Virol., 71: 7281-7288, 1997. Finke and Conzelmann, "Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3 'copy-back ambisense rabies virus ", J. Viral., 73: 3818-3825, 1999. Flamand and Bishop," Primary In Vivo Transcription of Vesicular Stomatitis Virus and Temperature-Sensitive Mutants of Five Vesicular Stomatitis Virus Complementation Groups ", J. of Virology, 12 (6): 1238-1252, 1973. Flamand and Pringle," The homologies of spontaneous and temperature-sensitive mutants of vesicular stomatitis virus isolated in chick embryos and BHK-21 cells ", J. Gen. Virol., 11: 81-85, 1971. Flanagan et al.," Rearrangement of the genes of vesicular stomatitis virus eliminates clinical disease in the natural host: new strategy for vaccine velopmenf, J. Virology, 75: 6107-6114,2001. Fredericksen and Whitt, "Attenuation of recombinant vesicular stomatitis viruses encoding mutant glycoproteins demónstrate to crifical role for maintaining a high pH threshold for membrane fusion in viral fitness", Virology, 240: 349-58, 1997. Gallione eí a /., "Nucleotide Sequences of the mRNA's encoding the Vesicular Stomatitis Virus N and NS Proteins ", J. Virol., 39: 529-636,1981. Gopalakrishna and Lenard, "Sequence alterations in temperature-sensitive M-protein mutants (complementation group lll) of vesicular stomatitis virus", Journal of Virology, 56 (3): 655-659,1985. Griffin, Measles Virus. In "Field's Virology", 3rd Edition, Lippincott-Raven, 1401-1442, 2001. Harty et al., "A Proline-Rich Motif within the Matrix Protein of Vesicular Stomatitis Virus and Rabies Virus Interacts with WW Domains of Cellular Proteins: Implications for Viral Budding ", J. Virology, 73 (4): 2921-2929, 1999. Holloway et al., "Isolation and characterization of temperature-sensitive mutants of vesicular stomatitis virus", Virology, 42 (4): 917-26,1970. Irie et al., "Functional Analysis of Late-Budding Domain Activity Associated with the PSAP Moif within the Vesicular Stomatitis Virus M Protein", J. Virology, 78 (14): 7823-7827, 2004. Jayakar and Whitt, "Identification of two additional products from the matrix (M) gene that contributes to vesicular stomatitis virus Cytopathology ", Journal of Virology, 76 (16): 8011-8018, 2002. Jayakar et al.," Mutations in The PPPY Motif of Vesicular Stomatitis Virus Matrix Protein Reduces Virus Budding by Inhibiting to Late Step in Virion Relase ", Journal of Virology, 74 (21): 9818-27, 2000. It will be sent to," The Membrane-Proximal Domain of Vesicular Stomatitis Virus G Protein Functions as a Membrane Fusion Potentiator and Can Induce Hemifusion ", Journal of Virology, 76 (23): 12300-11, 2002. Jeetendra eí al, The Membrane-Proximal Region of Vesicular Stomatitis Virus Glycoprotein G Ectodomain Is Critical for Fusion and Virus Infectivity", Journal of Virology, 77 (23): 12807-18, 2003. Kapikian et al., Am. J. Epidemiol. 4: 405-21, 1969. Kim et al., Am. J. Epidemiol. 4: 422-34, 1969. Li et al., "Site-specific mutations in vectors that express antigenic and temperature-sensitive phenotypes of the M gene of vesicular stomatitis virus", Journal of Virology, 62 (10): 3729-3737 , 1988. Lundh et al, "Non-Lethal Infection of Aminergic Reticulum Core Nuerons: Age-Dependent Spread of St Mutant Vesicular Stomatitis Virus from the Nose", Journal of Neuropathology and Experimental Neurology, 47 (5): 497-506, 1988. Morita eí al., "Phenotypic revertants of temperature-sensitive M protein mutants of vesicular stomatitis virus: sequence analysis and functional characterization", Journal of Virology, 61 (2): 256-263, 1987. Pringle et al, "Enhanced mutability associated with a temperature-sensitive mutant of vesicular stomatitis virus", Journal of Virology, 39 (2): 377-389, 1981. Pringle et al, "Isolation and characterization of temperature-sensitive mutants of vesicular Stomatitis virus, New Jersey serotype ", Journal of Virology, 8 (6): 836-841, 1971. Pringle, "Genetic Characteristics of Conditional Lethal Mutants of Vesicular Stomatitis Virus Induced by 5-Fluorouracii, 5-Azacytidine, and Ethyl Methane Sulfonate," Journal of Virology, 5 (5): 559-567,1970. Printz and Wagner, "Temperature-Sensitive Mutants of Vesicular Stomatitis Virus: Synthesis of Virus Specific Proteins", J. of Virology, 7 (5): 651-662, 1971. Rabinowitz et al., "Comparison of Central Nervous System Disease Produced by Wild-Type and Temperature-Sensitive Mutants of Vesicular Stomatitis Virus ", Infection and Immunity, 13 (4): 1242-1249, 1976. Rabinowitz et al.," The Uncoupled Relationship Between the Temperature-Sensitivity and Neurovirulence in Mice of Mutants of Vesicular Stomatitis Virus ", J. Gen. Virol, 35: 237-249, 1977. Roberts et al.," Vaccination with a Recombinant Vesicular Stomatitis Virus Expressing an Influenza Virus Hemagglutinin Provides Complete Protection from Influenza Virus Challenge " , Journal of Virology, 73: 3723-3732, 1999. Robinson and Whitt, "The Membrane-Proximal Stem Region of Vesicular Stomatitis Virus G Protein Confers Efficient Virus Assembly", Journal of Virology, 74 (5): 2239-2246,2000 Rose and Gallione, "Nucleotide Sequences of the mRNA's encoding the Vesicular Stomatitis Virus G and M Proteins Determined from cDNA Clones Containing the Complete Coding Regions", J. Virol, 39: 519 + 528, 1981. Rose ei a /., " An Effective Aids Vaccine Based on Live Atte nuated Vesicular Stomatitis Virus Recombinants ", Ce / 7, '106 (5): d39-49,2001. Rose et al., "Glycoprotein Exchange Vectors Based on Vesicular Stomatitis Virus Allow Effective Boosting and Generation of Neutralizing Antibodies to a Primary Isolate of Human Immunodeficiency Virus Type 1", Journal of Virology, 74 (23): 10903-10, 2000. Schlehuber and Rose, "Vesicular Stomatitis Virus (VSV) Recombinants Expressing the HIV-1 2F5 Epitope Within the VSV G Protein ", Abstract Number 321, AIDS Vaccine 2003, New York, September, 2003. Schlereth et al.," Successful Mucosal Immunization of Cotton Rats in the Presence of Measles-Specific Antibodies Depends on Degree of Attenuation of Vaccine Vector and Virus Dose ", Journal of General Virology, 84: 2145-2151, 2003. Schlereth et al.," Successful vaccine-induced seroconversion by single-immunization in the presence of measles virus -specific material antibodies ", J. Virology, 74: 4652-4657, 2000. Schnell et al.," Requirement for a Non-Specific Glycoprotein Cytoplasmic Domain Sequence to Drive Efficient Budding of Vesicular Stomatitis Virus ", The EMBO Journal, 17 (5): 1289-1296, 1998. Schnell et al., "Foreign glycoproteins expressed from recombinant vesicular stomatitis viruses are incorporated into virus particles", Proc. Nati Acad. ScL, USA, 93: 11359-11365, 1996. Wagner, "Pathogenicity and Immunogenicity for Mice of Temperature-Sensitive Mutants of Vesicular Stomafitis Virus", Infection and Immunity, 10 (2): 309-315.1974. Wertz et al., "Gene rearrangement attenuates expression and lethality of a nonsegmented negative strand RNA virus" Proc. Nati Acad. Sci. USA, 95: 3501-3506, 1998.

Claims (4)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A genetically modified vesicular stomatitis virus (VSV), characterized in that it comprises at least two different classes of mutations in its genome, the mutations selected from the group consisting of a temperature-sensitive mutation (ts), a point mutation, a gene-mixing mutation, a mutation Trunk G, a non-cytopathic M gene mutation, an ambisense RNA mutation, a truncated G gene mutation, an M gene insertion mutation, and a gene deletion mutation, where the two mutations synergistically attenuate the pathogenicity of the VSV .
2. 2. The VSV according to claim 1, further characterized in that the pathogenicity is further defined as neurovirulence.
3. 3. The VSV according to claim 1, further characterized in that the two mutations are a mutation of the truncated gene G and a mutation of the gene mix.
4. 4. The VSV according to claim 1, further characterized in that the G protein encoded by the truncated G gene has a deletion of the last twenty or twenty-eight carboxy-terminal amino acids. d.- The VSV according to claim 1, further characterized in that the G protein encoded by the truncated G gene has a cytoplasmic end domain consisting of one to nine amino acids. 6. The VSV according to claim 3, further characterized in that the N gene is mixed to 3'-PNMGL-5 'or 3'-PMNGL-d', relative to the 3'-NPMGL-d 'of the genome of the VSV of native cell, where N is the gene that encodes the nucleocapsid protein, P is the gene that encodes the phosphoprotein, M is the gene that encodes the matrix protein, G is the gene that encodes the binding glycoprotein and L is the gene that encodes RNA polymerase protein dependent RNA. 7 - The VSV according to claim 3, further characterized in that it comprises a mutated genome selected from the group consisting of S'-PNMG ^ -uL-d ', 3'-PNMG (ct-9) L-5' , 3'-PMNG (ct-i) L-5 'and 3'-PMNG (Ct-9) L-5', where N is the gene encoding the nucleocapsid protein, P is the gene encoding the phosphoprotein , M is the gene that encodes the matrix protein, G (Ct-i) is the gene that encodes the binding glycoprotein that has a deletion of the last twenty-eight carboxy-terminal amino acids, G (Ct-9) is the gene that encodes the binding glycoprotein that has the last twenty carboxy-terminal amino acids and L is the gene that encodes the RNA-dependent RNA polymerase protein. 8. The VSV according to claim 7, further characterized in that it comprises a third class of mutation in its genome, wherein the mutation is a ts mutation, an ambisense RNA mutation, a mutation of the non-cytopathic M gene, a mutation of deletion of the gene, an insertion mutation of the gene or a point mutation. 9. The VSV according to claim 1, further characterized in that the VSV injected intracranially in female Swiss-Webster mice of 4 weeks of age has an LD50, 100 times, 1000 times, 10000 times or 100 000 times greater than the VSV of native type injected intracranially in female Swiss-Webster mice 4 weeks of age. 10. The VSV according to claim 1, further characterized in that the two mutations are a mutation of the truncated G gene and a mutation of the non-cytopathic M gene. 11. The VSV according to claim 10, further characterized in that the non-cytopathic mutation (M (nc)) of the M gene is a mutation in methionine 33 and methionine 51 of the M gene. 12.- The VSV according to the claim 10, further characterized in that it comprises a mutated genome of 3'-NPM (ncp) G (Ct-i) Ld 'or 3'-NPM (ncP) G (ct-g) L-5'. 13. The VSV according to claim 1, further characterized in that the two mutations are mutations of the gene (N (tS)) and a mutation of the gene (L (tS)). 14. The VSV according to claim 13, further characterized in that it comprises a mutated genome of 3'-N (tS) PMGL (ts) -5 '. 1 d.- The VSV according to claim 1, further characterized in that a mutation class is a G trunk mutation. 16. - A genetically modified VSV vector comprising the VSV according to claim 1, further characterized in that it comprises at least one foreign RNA sequence inserted into or replacing a region of the VSV genome not essential for replication. 17. The vector according to claim 16, further characterized in that the foreign RNA is also defined as an open reading structure (ORF). 18. The vector according to claim 16, further characterized in that the foreign RNA is selected from the group consisting of an HIV gene, an HTLV gene, a SIV gene, an RSV gene, a gene PIV, an HSV gene, a CMV gene, an Epstein-Barr virus gene, a virus gene Varicella-Zoster, a mumps virus gene, a measles virus gene, an influenza virus gene, a poliovirus gene, a rhinovirus gene, a hepatitis A virus gene, a hepatitis virus gene B, a hepatitis C virus gene, a Norwaik virus gene, a togavirus gene, an alpha virus gene, a rubella virus gene, a rabies virus gene, a Marburg virus gene, a virus gene of Ebola, a papilloma virus gene, a polyoma virus gene, a metapneumovirus gene, a coronavirus gene, a Vibrio cholera gene, a Streptococcus pneumoniae gene, a Streptococcus pyogenes, a gene of Helicobacter pylori, a gene of Streptococcus agalactiae, a gene of Neisseria meningittidis, a gene of Neisseria gonorrheae, a Corynebacteria diphtheriae gene, a Clostridium tetani gene, a Bordetella pertussis gene, a Haemophilus gene, a Chlamydia gene, an Escherichia coll gene, a gene that codes for a cytokine, a gene that encodes the epitope helper T, a gene encoding a CTL epitope, a gene encoding an adjuvant and a gene encoding a co-factor. 19. The vector according to claim 18, further characterized in that the HIV gene is selected from the group consisting of gag, env, pol, vif, nef, tat, vpr, rev, or vpu. 20. The vector according to claim 19, further characterized in that the HIV gene is gag and the mutated genome is 3'-gag PNMG ^ DL-d ', 3'-gag? -PNMG (ct-9) L -5 ', 3'-gagrPMNG (ct-i) L-5', 3'-gagr PMNG (ct-9) L-5 ', 3'-PNMG (CM) L-gag5-d', 3'-PNMG (Ct-9) L-gag5-d ', S'-PMNG ^ D -gag5- d 'or 3'-PMNG (ct-9) L-gag5-5'. 21. An immunogenic composition comprising an immunogenic dose of a genetically modified VSV vector according to claim 16. 22. A method for immunizing a mammalian host against bacterial infection, characterized in that it further comprises administering an immunogenic dose of a genetically modified VSV vector comprising: a) at least two different classes of mutations in its genome, the mutations are selected from the group consisting of a ts mutation, a point mutation, a gene blending mutation, a G stem mutation , a non-cytopathic M gene mutation, an ambisense RNA mutation, a truncated G gene mutation, a G gene insertion mutation, and a gene deletion mutation, wherein the two mutations synergistically attenuate the pathogenicity of the VSV and ( b) at least one foreign RNA sequence inserted into or replacing a region of the VSV genome not essential for replicing ation, wherein the RNA encodes a bacterial protein selected from the group consisting of a protein of Vibrio cholera, a protein of Streptococcus pneumoniae, a protein of Streptococcus pyogenes, a protein of Streptococcus agalactiae, a protein of Helicobacter pylori, a protein of Neisseria meningitidis, a protein of 0 Neisseria gonorrheae, a protein of Corynebacteria diphtheria, a protein of Clostridium tetani, an Bordetella pertussis protein, a Haemophilus protein, a Chlamydia protein and an Escherichia coli protein. 23. A method for immunizing a mammalian host against viral infection, further characterized in that it comprises administering an immunogenic dose of a genetically modified VSV vector, which comprises: (a) at least two different classes of mutations in its genome, the mutations are selected from the group consisting of a ts mutation, a point mutation, a gene-blending mutation, a G-stem mutation, a mutation of non-cytopathic M gene, an ambisense RNA mutation, or a truncated G gene mutation, a G gene insertion mutation, and a gene deletion mutation, wherein the two mutations synergistically attenuate the pathogenicity of the VSV and (b) at least one foreign RNA sequence inserted into or replacing a region of the VSV genome not essential for replication, wherein the RNA encodes a viral protein selected from the group consisting of an HIV protein, an HTLV protein, a SIV protein, an RSV protein, a PIV protein, an HSV protein, a CMV protein, an Epstein-Barr virus protein, a Varicella-Zoster virus protein, a mumps virus protein, a protein the measles virus, an influenza virus protein, a poliovirus protein, a rhinovirus protein, a hepatitis A virus protein, a hepatitis B virus protein, a protein of the hepatifis C virus, a protein of the Norwaik virus, a togavirus protein, an alphavirus protein, a rubella virus protein, a rabies virus protein, a Marburg virus protein, an Ebola virus protein, a papilloma virus protein, a protein of the polyoma virus, a metapneumovirus protein and a coronavirus protein.