ES2345198T3 - IMMUNOLOGICAL COMPOSITION AND PROCEDURES. - Google Patents

Abstract

Use of a first composition comprising a DNA plasmid comprising a DNA sequence encoding an antigen under the control of regulatory sequences that direct its expression by said DNA plasmid in the preparation of a medicament to induce a specific immune response. of the antigen in a mammalian subject, wherein the induction of said antigen-specific immune response comprises (a) administering to said subject an effective amount of said first composition; and (b) administering to said subject an effective amount of a second composition comprising a recombinant vesicular stomatitis virus (VSV) comprising a nucleic acid sequence encoding said antigen under the control of regulatory sequences that direct its expression by said recombinant VSV.

Description

Composition and immunological procedures.

Background of the invention

To improve the efficacy of immunogenic compositions a variety of immunogenic compositions and methods using protein compositions, plasmid-based compositions and recombinant virus constructs as immunogenic compositions have been described. Previous studies have shown that plasmid-based immunogenic compositions, after systemic application, favor the systemic immune system for a second systemic immunization with a traditional antigen, such as a protein or a recombinant virus (see, for example, Xiang et al., 1997 Springer Semin. Immunopathol ., 19: 257-268; Schneider, J. et al., 1998 Nature Med., 4: 397; and Sedeguh, M. et al., 1998 Proc. Natl. Acad. Sci., USA , 95: 7648; Rogers, WO et al., 2001 Infec. & Immun ., 69 (9): 5565-72; Eo, SK, et al., 2001 J. Immunol., 166 (9): 5473-9 ; Ramshaw IA and Ramsay, AJ, 2000 Immunol. Today, 21 (4): 163-5). For a review of known vaccine delivery systems with respect to HIV, see Sutter et al., 2001 AIDS 15 (supplement 5): 139-145.

A frequently used live vector DNA / stimulus primer regimen involves vaccine viruses for the stimulus. Examples in the recent literature include immunization for human immunodeficiency virus (HIV) (Hanke T. et al., 2002 Vaccine 20 (15): 1995-8; Amara RR et al., 2002 Vaccine 20 (15): 1949-55 ; Wee EG et al., 2002 J. Gen. Virol. , 83 (Pt 1): 75-80; Amara RR et al., 2001 Science 292 (5514): 69-74). It has been described that immunizations with primer-stimulation with DNA and modified vaccine virus vectors expressing antigens such as herpes simplex virus type 2 virus , Leishmania infantum P36 / LACK antigen, Plasnaodium falciparuna TRAP antigen, HIV / VIS antigens , murine tuberculosis antigens, and influenza antigens, give rise to specific antibody and cytokine responses (see, for example, Meseda CA et al., 2002 J. Infect. Dis. , 186 (8): 1065-73; Amara RR et al., 2002 J. Virol. , 76 (15): 7625-31; Gonzalo RM et al., 2002 Vaccine , 20 (7-8): 1226-31; Schneider J. et al., 2001 Vaccine , 19 (32): 4595-602; Hel Z. et al., 2001 J. Immunol. , 167 (12): 7180-91; McShane H. et al., 2001 Infec. & Immun. , 69 (2) : 681-6 and Degano P. et al 1999 Vaccine , 18 (7-8): 623-32 influenza and malaria models).

It has recently been described that genetic immunization regimens with priming-stimulation with plasmid adenovirus induce immunity against specific alpha-fetoprotein tumor and protect the pig from classical swine fever (see, for example, Meng WS 2001 Cancer Res. , 61 ( 24): 8782-6; Hammond, JM et al., 2001 Vet. Microbe. , 80 (2): 101-19; and U.S. Patent No. 6,210,663).

Other plasmid priming regimens of DNA-stimulus with virus have been described. See, for example, Matano T. et al., 2001 J. Virol. , 75 (23): 11891-6 (a DNA prime / Sendai virus vector boost). Priming of DNA with recombinant poxvirus stimulation has been described for treatment of HIV-1 (see, for example, Kent, SJ et al., 1998 J. Virol. , 72: 10180-8; Robinson, HL et al., 1999 Nat. Med. , 5: 526-34; and Tartaglia, J. et al., 1998 AIDS Res. Human Retrovirus ., 14: pages 291-8).

Recombinant VSV vectors have been described in Haglund et al., 2002 Journal of Virology 76 (6): 2730-2738, and WO 96/34625 and WO 02/097091. Although a plurality of priming regimens with DNA / viral stimulation are being evaluated, the immunization regimens currently described have several disadvantages. For example, some of the viruses indicated above cause disease symptoms in subjects; others may result in recombination in vivo . Still other viruses are difficult to prepare and / or have a limited ability to accept foreign genes. Still other viruses have disadvantages caused by significant pre-existing vector immunity in man, and other safety implications.

The need remains in the technique of new and useful immunization regimens that can generate greater  levels of cellular and humoral immune responses to antigens in question and meet safety requirements, ease of preparation and the ability to overcome the natural immune response of mammalian hosts for vectors after immunization with reinforcers

Summary of the invention

The present invention provides a new procedure, composition and kit for induction in a mammalian subject of an immune response against a pathogenic antigen or another antigen by a primed / stimulus regime that shows a surprising synergistic stimulation of cellular immune response against the antigen compared to the results obtained well with the plasmid component of DNA or with the viral component recombinant, when administered individually.

In one embodiment the invention provides a new procedure for inducing a specific immune response of the antigen in a mammalian subject. The procedure involves the administration to the subject of an effective amount of a first composition comprising a DNA plasmid comprising a DNA sequence encoding an antigen under the control of regulatory sequences that direct its expression in a mammalian cell by the plasmid DNA. The procedure it also implies the administration to the subject of an effective amount of a second composition comprising a stomatitis virus recombinant vesicular (VSVr) comprising an acid sequence nucleic encoding the antigen under sequence control regulators that direct its expression in the cell of mammal by the VSVr. In one embodiment the recombinant VSV It is a competent replication virus, attenuated. In another embodiment Recombinant VSV is a non-replicating virus. Administrations of the first and second composition can be in any order. In addition, the invention contemplates multiple administrations of one of the compositions followed by multiple administrations of the other composition. In one embodiment it is co-administered. preferably a cytokine.

In another embodiment the invention provides a immunogenic composition to induce an immune response antigen specific to an antigen in a mammalian subject. The immunogenic composition comprises a first composition that comprises a DNA plasmid comprising a DNA sequence that encodes the antigen under the control of regulatory sequences that direct expression thereof by the DNA plasmid. This composition also includes at least one replication component, recombinant vesicular stomatitis virus (VSV) comprising a nucleic acid sequence encoding the same low antigen the control of regulatory sequences that direct the expression of same by the recombinant VSV.

In yet another embodiment the invention provides a kit for use in a therapeutic or prophylactic procedure of induction of a higher level of specific immune response of the antigen in a mammalian subject. The kit includes, among others, the minus a first composition comprising a DNA plasmid that comprises DNA sequence encoding an antigen under control of regulatory sequences that direct its expression in a mammalian cell; at least a second composition that It comprises a replication component, stomatitis virus recombinant vesicular (VSVr) comprising an acid sequence nucleic encoding said antigen under sequence control regulators that direct its expression in said cell of mammal, and instructions to implement the procedure above.

In another embodiment the invention provides the use of the immunogenic composition described above or components thereof in the preparation of a medicament for induction of an immune response in an animal against the antigen Used in the composition.

Other aspects and embodiments of this Invention are described in the following detailed description.

Brief description of the drawings

Figure 1A is a schematic diagram of an illustrative plasmid DNA encoding a simian immunodeficiency virus (VIS) gag p37 protein. The diagram shows that the plasmid contains a human cytomegalovirus promoter / enhancer (CMVH) that drives the expression of the gag protein, a polyadenylation site of bovine growth hormone, (BGH polyA), an origin of the replication sequence ( ori) and a kanamycin resistance marker gene ( kan R ).

Figure 1B is a schematic diagram of an illustrative bicistronic plasmid DNA encoding the two subunits p35 and p40 of rhesus interleukin 12. The p35 subunit is under the control of the CMVH promoter and has a SV40 poly A site. The p40 subunit is under the control of the simian cytomegalovirus (CMVS) promoter and has a poly A BGH site, and is transcribed into the reverse direction. This plasmid also contains an ori sequence and a kan R gene.

Figure 2 is a bar chart showing ELISpot answers for interferon gamma VSV N-specific (IFN- γ) in cells unfractionated peripheral blood mononuclear (PBMC) in animals immunized with a priming / stimulus regime of the present invention. The leftmost dark bars represent a immunization protocol with the DNA plasmid encoding the VIS gag protein with a stimulation of a VSV vector that expresses a hemagglutinin protein (flu HA) from the flu. Gray bars clear represent a protocol of the invention that involves a plasmid immunization based on priming gag DNA followed by a VSV stimulus that expresses HIV gag and env proteins. The clear bars represent a protocol that involves immunization priming with an empty or control DNA plasmid (DNA with) followed by immunization with a VSV that expresses gag proteins and HIV env. The rightmost dark bars represent a protocol that involves priming immunization with plasmid DNA followed by immunization with a VSV expressing flu HA protein. Each group represents results from 5 animals.

Figure 3 is a bar chart showing ELISpot responses to interferon gamma (IFN- γ) specific for HIV env 6101 in Unfractionated PBMC of animals immunized with a regimen of priming / stimulation of the present invention. Light gray bars more to the left represent an immunization protocol with the DNA plasmid encoding the VIS gag protein with a stimulus of a VSV vector expressing the flu HA protein. The bars to stripes represent a protocol of the invention that involves a plasmid immunization based on priming gag DNA followed by a VSV stimulus that expresses HIV gag and env proteins. The checkered bars represent a protocol that involves a priming immunization with an empty control DNA followed by immunization with a VSV that expresses HIV gag and env proteins. Dot bars represent a protocol that implies a priming immunization with control DNA plasmid followed by immunization with a VSV stimulus that expresses the flu HA protein. Each group represents results from 5 animals. Asterisks indicate where there were statistically significant differences to p = 0.0001.

Figure 4 is a graph showing anti-VIS gag p27 antibody titers in serum by enzyme-linked immunosorbent assay (ELISA) for animals immunized with a primed / stimulus regime of the present invention. Plasmid DNA was administered on day 0, week 4 and week 8 and VSV stimuli (Indiana G serotype) and VSV (Chandipura G serotype) were administered at week 15 and 23, respectively. An immunization protocol with the DNA plasmid encoding the VIS gag protein is depicted with a stimulation of a VSV vector expressing flu HA protein with (black). A protocol of the invention is presented that involves an immunization with plasmid of priming gag DNA followed by a VSV stimulus that expresses HIV gag and env proteins with (blacksquare). A protocol is represented that involves priming immunization with an empty control DNA followed by immunization with a VSV that expresses HIV gag and env proteins with (115 {115}). A protocol is depicted that involves priming immunization with control DNA plasmid followed by immunization with a VSV expressing the flu HA protein with (?). Each group represents results from 5 animals. Statistically significant differences between groups are shown as p = 0.0073 (*); p = 0.5941 (#) or p = 0.0027 ( 100 ).

Figure 5 is a graph showing spots SIV gag-specific per million cells evaluated by ELISpot assay for animals immunized with a regimen of priming / stimulation of the present invention. DNA was administered from plasmid on day 0, week 4 and week 8 and were administered VSV stimuli (Indiana G serotype) and VSV (Chandipura G serotype) in week 15 and 23, respectively. A protocol of immunization with the DNA plasmid encoding the gag protein of VIS with a stimulus of a VSV vector expressing flu HA protein with (\ blacklozenge). A protocol of the invention is presented which involves an immunization with plasmid DNA priming gag followed of a VSV stimulus that expresses HIV gag and env proteins with (\ blacksquare). It represents a protocol that implies a priming immunization with an empty control DNA followed by immunization with a VSV that expresses HIV gag and env proteins with (\ ding {115}). The (?) Represents a protocol that involves priming immunization with control DNA plasmid followed by immunization with VSV expressing flu HA protein. Every group represents results of 5 animals. Differences are shown statistically significant between groups with parentheses for p = 0.0001 and p = 0.0002.

Figure 6 is a graph that shows that high immune responses facilitated by combinations of priming / stimulus indicated by the same symbols as in figure 5 result in greater protection against AIDS, measured by a loss reduced CD4 T cells in days after exposure.

Figure 7 is a graph that shows that high immune responses facilitated by combinations of priming / stimulus indicated by the same symbols as in figure 5 result in greater protection against AIDS, measured by a reduction in circulating plasma virus (virus copies / ml) in days after exposure

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Detailed description of the invention

The invention relates to a new method of induction of an antigen specific immune response in a mammalian or vertebrate subject using certain combinations components of immunogenic compositions described in the art previous, and optimizing the components to produce results Amazing and synergistic. In general, the procedure involves administration to the subject of an effective amount of a composition  which includes a DNA plasmid comprising a DNA sequence which encodes an antigen under the control of regulatory sequences which direct its expression in a mammalian cell or vertebrate by the plasmid DNA. The procedure also includes a stage of administration to the subject of an amount effective of a composition comprising a component of replication, recombinant vesicular stomatitis virus (RSVV). This VSVr comprises a nucleic acid sequence encoding the antigen under the control of regulatory sequences that direct the expression thereof in the mammalian or vertebrate cell by of the VSVr.

The first of these two compositions immunogenic to be administered in order is referred to as the priming composition. The second of these two compositions immunogenic to be administered in order is called the stimulus composition Therefore, well the DNA composition is can administer as the priming composition and the composition of VSV vector is administered as the stimulus composition or vice versa. In one embodiment the priming composition is administered to the subject at least once or multiple times before administration of the stimulus composition. After this, the composition of stimulus is subsequently administered to the subject at least once or multiple times. In addition the invention contemplates multiple administrations of one of the compositions followed by multiple administrations of the other composition. The procedure contemplates in addition the administration of an effective amount of a cytokine as a stage in the procedure.

It has been surprisingly found that the practice of this procedure induces in a mammalian subject a immune response that includes an increase in T-cell response CD8 + against the antigen greater than that achieved with the administration of recombinant DNA or VSV plasmid alone. From done, as indicated in the following examples, the answer immune is greater than expected with an additive combination of two immunogenic compositions. Therefore the immune response induced by the new procedure is a drastic increase and synergistic in cellular and / or antibody responses against antigen. See, for example, table 1 below and the figures 4 and 5, in which the specific cellular response of the peak antigen against the gag of HIV-1 is more than 3 times higher that the response to the administration of an immunogenic composition  plasmid DNA alone or almost 9 times greater than the response to the administration of the immunogenic composition of VSVr alone.

Although it is contemplated that the mammalian subject is a primate, preferably a human being, the invention is not limited to the identification of the mammalian subject. The components of This procedure is described in more detail below and in reference to the cited documents that are incorporated as reference to provide details known to a specialist in the technique

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A. Immunogenic composition of plasmid DNA

Immunogenic compositions of the present invention include a DNA plasmid comprising a sequence of DNA encoding a selected antigen for which it is desired an immune response In the plasmid the selected antigen is under the control of regulatory sequences that direct expression thereof in a mammalian or vertebrate cell. The components of the plasmid itself are conventional.

Non-viral plasmid vectors, useful in this invention contain isolated and purified DNA sequences that comprise DNA sequences encoding the immunogenic antigen selected. The DNA molecule can be derived from species viral or non-viral, for example, bacterial species that have been designed to encode an exogenous nucleic acid sequence or heterologous Such plasmids or vectors may include sequences of virus or phage A variety of non-viral vectors are known in the technique and may include, without limitation, plasmids, vectors bacterial, bacteriophage vectors, "naked" DNA and DNA condensed with cationic lipids or polymers.

Examples of bacterial vectors include, but are not limited to, sequences derived from the bacillus Calmette Guérin (BCG), Salmonella, Shigella, E. coli, and Listeria , among others. Suitable plasmid vectors include, for example, pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pK37, pKC101, pAC105, pVA51, pKH47, pUB110, p259, p259, p259, p259 pBR313, pML21, RSF2124, pCRl, RP4, pBAD18, and pBR328.

Examples of suitable inducible Escherichia coli expression vectors include pTrc (Amann et al., 1988 Gene , 69: 301-315), arabinose expression vectors (eg, pBAD18, Guzman et al., 1995 J. Bacteriol. , 177: 4121-4130), andpETIId (Studiery col., 1990 Methods in Enzymology , 185: 60-89). The pTrc vector target gene expression relies on the transcription of RNA polymerase into a host of a hybrid trp-lac fusion promoter. The expression of the pETIId vector target gene relies on the transcription of a T7 gn10-lac fusion promoter mediated by a co-expressed viral T7 gn1 RNA polymerase. This viral polymerase is supplied by BL21 (DE3) or HMS 174 (DE3) host strains of a resident profago that protects a T7 gn1 gene under the transcriptional control of the lacUV5 promoter. The pBAD system rests on the inducible arabinose promoter that is regulated by the araC gene. The promoter is induced in the presence of arabinose.

The promoter and other regulatory sequences that conduct antigen expression in the mammalian host or desired vertebrate can be similarly selected from a extensive list of promoters known to be useful for that purpose. A variety of these promoters are described below. In a embodiment of the immunogenic DNA plasmid composition described below, useful promoters are the human cytomegalovirus (CMVH) promoter / enhancer (described, for example, in U.S. Patent Nos. 5,168,062 and 5,385,839) and the CMVS enhancer promoter.

Additional regulatory sequences for inclusion in a nucleic acid sequence, molecule or vector of this invention include, without limitation, a sequence enhancer, a polyadenylation sequence, a sequence splice donor, and a splice acceptor sequence, a site for transcription initiation and termination positioned at beginning and end, respectively, of the polypeptide to be translate, a ribosome binding site for translation in the transcribed region, an epitope tag, a sequence of nuclear location, an IRES element, a "TATA" element of Goldberg-Hogness, an enzyme cleavage site restriction, a selectable marker and the like. Sequences enhancers include, for example, the tandem repeat of 72 SV40 DNA bp or retroviral terminal repeats or LTRs, etc. and are used to increase transcriptional efficiency.

These other useful components in plasmids of DNA, including, for example, origins of replication, sequences polyadenylation (for example, BGH polyA, SV40 polyA), markers of drug resistance (e.g., kanamycin resistance) and similar can also be selected among sequences widely known, including those described in the examples and cited specifically below.

The selection of promoters and other common vector elements are conventional and many such sequences are available with which to design the plasmids useful in this invention. See, for example, Sambrook et al., Molecular Cloning A Laboratory Manual , Cold Spring Harbor Laboratory, New York, (1989) and references cited in this document, for example, on pages 3.18-3.26 and 16.17-16.27 and Ausubel and col., Current Protocols in Molecular Biology , John Wiley & Sons, New York (1989). All plasmid components useful in this invention can be readily selected by one skilled in the art from materials known in the art and are available in the pharmaceutical industry. The selection of plasmid components and regulatory sequences is not considered a limitation of this invention.

Examples of DNA plasmid constructs suitable for use in immunogenic compositions are described in detail in the following patent publications, by example, international patent publications numbers WO98 / 17799, WO99 / 43839 and WO98 / 17799; and United States patents numbers 5,593,972; 5,817,637; 5,830,876; and 5,891,505, among others.

Similarly, the selected antigen may be an antigen identified in the following description. In one embodiment of the immunogenic compositions in this invention, the selected antigen is an HIV-1 antigen, such as one expressed by gag, pol, env, nef, vpr, vpu, vif and tat . Preferably the antigen sequence and other components of the DNA plasmid are optimized, such as by codon selection appropriate to the intended host and by elimination of any inhibitory sequence, also described below with respect to antigen preparation.

This immunogenic composition may include by both a plasmid that encodes a simple selected antigen for expression in the host. In accordance with the present procedure the Plasmid composition also comprises a DNA plasmid that comprises a DNA sequence that encodes more than one copy of the Same antigen selected. Alternatively, the composition may contain a plasmid expressing selected antigens multiple. Each antigen can be under the control of elements or separate regulatory components. Alternatively each antigen may be under the control of the same elements Regulatory In yet another embodiment the plasmid composition of DNA can contain multiple plasmids, in which each plasmid of DNA encodes the same or a different antigen.

In another additional embodiment, the composition plasmid immunogenic DNA may also contain, as a plasmid component of individual DNA or as part of the plasmid of DNA that contains the antigen, a nucleotide sequence that encodes a cytokine, lymphokine or other genetic adjuvant desirable. A host of such are identified below. suitable adjuvants for which they are available nucleic acid sequences. In the exemplified embodiments in this invention, a desirable cytokine for administration with The DNA plasmid composition of this invention is interleukin-12.

The DNA plasmid composition is administered. Desirably in a diluent, excipient or vehicle Pharmaceutically acceptable, such as those described below. While the composition can be managed by any route selected from administration, in one embodiment a procedure desirable administration is co-administration via intramuscular of a composition comprising plasmids with bupivacaine as the facilitating agent, described to continuation.

In an embodiment of the process of the present invention in which the DNA composition is the priming composition, the method includes the administration of at least one plasmid of DNA before the RSVV, the plasmid comprising a sequence encoding an antigen for which it is desired to induce an immune response. In another embodiment the DNA priming composition further comprises a second plasmid encoding a selected cytokine. In yet another embodiment, exemplified below, the DNA priming composition includes three plasmids, a plasmid expressing a first antigen, the second plasmid expressing the second different antigen and a third plasmid expressing a selected cytokine adjuvant. As detailed in the examples, one embodiment of a DNA priming composition contains three optimized plasmids, (1) a plasmid encoding an RNA optimized VIS gag p37 gene (see Figure 1A); (2) a plasmid encoding the two p35 and p40 subunits of rhesus IL-12, under the individual control of two promoters (see Figure 1B) and (3) a plasmid encoding the HIV-1 gp160 env gene.

When used as a priming composition This DNA plasmid composition is administered once or preferably more than once before the composition of VSVr of stimulus. When used as the stimulus composition is Plasmid DNA composition is administered once or preferably more than once after administration of the VSVr priming composition.

B. VSVr immunogenic composition

Another immunogenic composition useful in the methods and compositions of this invention is a live attenuated vesicular stomatitis virus (VSV) release vehicle, competent for replication. This recombinant VSV comprises a nucleic acid sequence that encodes the selected antigen under the control of regulatory sequences that direct its expression in the mammalian or vertebrate cell. Recombinant VSV vectors have been described in Haglund et al., 2002 Journal of Virology 76 (6): 2730-2738, and WO 96/34625 and WO 02/097091. In the methods of the present invention the antigen used in the DNA plasmid composition is the same antigen used in the VSVr immunogenic composition.

VSV is a cattle virus, which is a member of the taxonomic order designated mononegavirals, and comprises a non-segmented, negative strand RNA genome of 11 kb that encodes four internal structural proteins and one outer transmembrane protein. In the order 3 'to 5' the genes encode proteins called the nucleocapsid (N), phosphoprotein (P), matrix protein (M), transmembrane glycoprotein (G) and polymerase (L). A live VSV can be isolated and "rescued" using techniques known in the art. See, for example, United States patents numbers 6,044,886; 6,168,943; and 5,789,299; International Patent Publication No. WO 99/02657; Conzelmann, 1998, Ann. Rev. Genet. , 32: 123-162; Roberts and Rose, 1998, Virol. , 247: 1-6; Lawson et al., 1995 Proc. Natl Acad. Sci. , USA 92: 4477-4481. Additionally, for example, the VSV genomic sequence (Indiana) is established with the access number NC001560 in the NCBI database. Other VSV sequences are available, including VSV (Chandipura) sequences in that database; for example see access numbers Ay382603, Afl28868, V01208, V01207, V01206, M16608, M14715, M14720 and J04350, among others. VSV strains, such as New Jersey and Indiana, among others, are also available in tanks such as the American Type Culture Collection, Rockville, Maryland (see, for example, access numbers VR-1238 and VR-1239). Other sequences are described or referred to in the documents cited throughout this specification.

VSV genomes have been shown to accommodate to more than one strange gene, with expansion to at least three kilobases The genomes of these viruses are very stable, they do not suffer recombination and rarely incur significant mutations. Also because its replication is cytoplasmic, and its genomes are constituted by RNA, are unable to integrate within genomes of infected host cells. Also these viruses from  Negative strand RNAs possess transcriptional control sequences relatively simple, which are easily manipulated to Efficient foreign gene insertion. Finally, the level of strange gene expression can be modulated by changing the position of the foreign gene regarding the viral transcription promoter. He 3 'to 5' gradient of gene expression reflects the probability decreasing that transcription of viral polymerase will successfully hinder each gene start / end gene signal intergenic found when it progresses along the tempering of the genome Therefore strange genes are abundantly expressed arranged in proximity to a promoter initiating the 3 'terminal transcription, while those inserted in more distal genomic positions will do less.

The VSV is replicated with a high level of titles in a large set of different cell types and express themselves viral proteins in great abundance. This does not only mean that the VSV will act as a functional foreign gene release vehicle powerful, but they can also be scaled to levels of manufacture of relevant VSVr vectors in cell lines approved for the production of human biological products.

The VSVr has a remarkable ability to release foreign genes that encode critical protective immunogens of viral pathogens for a broad group of different cell types, and subsequently cause abundant expression of authentically configured immunogenic proteins (Haglund, K., et al., 2000 Virol. , 268: 112-21; Kahn, JS et al., 1999 Virol. , 254: 81-91; Roberts, A. et al., 1999 J. Virol. , 73: 3723-32; Rose, NF et al., 2000 J. Virol. , 74: 10903-10; and Schlereth, B. et al. 2000 J. Virol. , 74: 4652-7). Immunogens, thus expressed, simultaneously give rise to both long-lasting virus neutralizing antibody responses, as well as protective cytotoxic T lymphocytes (CTL) (Roberts, A. et al., 1998 J. Virol., 72: 4704- eleven).

In addition to the efficacy of the RSVV, this live virus gene release vehicle is safe, because the wild-type VSV produces few or no symptoms of disease or pathology in healthy humans, even in the case of substantial virus replication ( Tesh, RB et al., 1969 Am. J. Epidemiol. , 90: 255-61). Additionally, human infection with, and therefore pre-existing VSV immunity is rare. Given the additional attenuation these VSVr compositions are suitable for use in human individuals less with compromised immunity or less robust. A significant advantage of using the VSV vector in this procedure is that there are a number of VSV serotypes due to the exchange or modification of the VSV viral binding G protein. Therefore, different VSV vector serotypes carrying the same heterologous antigen can be used for repeated administration avoiding any interfering neutralizing antibody response generated for VSV G protein by the host's immune system.

A recombinant VSV can be designed using techniques previously described in the art, which carries the antigen selected and its regulatory sequences inserted in any VSV position under the control of the transcription promoter viral. In one embodiment the heterologous gene encoding the antigen selected is inserted between the coding regions G and VSV L In another embodiment the heterologous gene can be fused in  the protein G site. Still in other embodiments the gene heterologous merges with the site, or is adjacent to, any of the other genes of VSV.

Even in other embodiments the genes are "randomized" at different positions in the genome. In particular, the N gene is "randomized" at different positions in the genome. Cloning to produce the randomized recombinant cDNA sequences involves modification of the original VSV plasmid skeleton (pVSV-XN1). Three variants of this original vector include plasmids that deviate from the order of the normal gene ( 3'-N-PMGL-5 ' ) as follows: i) 3'- PNMGL -5'; ii) 3'- PMNGL -5 '; and iii) 3'- PMGNL -5 '. The cloning strategy used to create these plasmids uses a procedure described by Ball, LA et al. 1999 J. Virol. , 73: 4705-12. This technique has the advantage of the fact that the gene end / gene start signals found between each coding sequence are almost identical, and allow gene redispositions to be constructed without introducing any nucleotide substitution. Alternatively, strategic point mutations can be introduced in non-coding sequences to create convenient restriction sites that facilitate redisposition of the
genome

Even in additional embodiments of these vectors the coding sequence of the carboxy terminal for the 29 amino acid cytoplasmic domain of the G gene is truncated by amino acid deletion from the 5'C terminus of the G gene. alternatively the G gene is completely deleted. In a embodiment the entire cytoplasmic domain of the G gene is eliminated. In another embodiment at least 28 amino acids are removed from the domain cytoplasmic Even in a further embodiment they are delighted approximately 20 amino acids of the cytoplasmic domain. Still in one additional embodiment approximately 10 or less are deleted amino acids of the cytoplasmic domain.

Both the randomized genome approach and the G protein modification approach generate partial growth defects (Flanagan, EB, et al. 2001 J. Virol . 75: 6107-14; Schnell, MJ et al. 1998 EMBO J. , 17: 1289-96). It is concluded that such modifications of the VSV can lead to a more attenuated phenotype or even a non-replicating VSV for use in different embodiments of the present invention. See, for example, Johnson, JE et al., 1998 Virol. , 251: 244-52.37; Johnson, JE, et al., 1997 J. Virol. , 71: 5060-8.

Similarly, the selected antigen may be an antigen identified in the following description. In one embodiment of the immunogenic compositions of this invention, the selected antigen is a (gp 160) gag and / or env HIV-1 gene of clade B virus isolate. In still other embodiments the antigen is a pol, nef gene. , vpr, vpu, vif or tat of HIV-1. Preferably the antigen sequence is optimized such as by codon selection appropriate to the intended host and / or by removal of any inhibitory sequence, also described below in relation to antigen preparation.

To overcome any potential problem of reduced vector replication efficiencies with administration sequential, a vector set of similar design, which carries each a G gene of a different VSV serotype allows immunizations of successful stimulus. The primary amino acid sequences of G proteins from VSV Indiana, New Jersey, and Chandipura, are divergent enough for pre-existing immunity to one does not alter the infection and replication of the others. Therefore the neutralizing antibody response generated by VSVr (Indiana) should not interfere with the replication of VSVr (New Jersey) or of VSVr (Chandipura). A vector set can be prepared that may allow successful sequential immunizations by replacing the VSV Indiana G gene with any of VSV divergent homologue Chandipura or VSV New Jersey, which form three vectors immunologically different.

Said VSVr immunogenic composition may therefore include a VSVr encoding a selected antigen unique for expression in the host. In accordance with this procedure, the immunogenic composition of VSVr comprises a VSVr comprising a nucleic acid sequence that encodes more of a copy of the same selected antigen. Alternatively The composition may contain a VSVr that expresses multiple selected antigens. Each antigen can be under control of separate regulatory elements or components. So alternatively each antigen may be under their control regulatory elements In yet another embodiment the composition of VSVr can contain multiple VSVr, in which each VSVr encodes The same or a different antigen.

In another additional embodiment the composition VSVr immunogenic may additionally contain or be administered with a cytokine, lymphokine or genetic adjuvant. Cytokine can be administered as a protein or in a plasmid as cited above or can be encoded by insertion of the sequence that encodes the cytokine in a VSV recombinant For example, the sequence that encodes the cytokine is can insert at any position in the VSV genome and express from the viral transcription promoter. Be identify a host of such adjuvants below suitable for which sequences of nucleic acids. In the embodiments exemplified in this invention a desirable cytokine for administration with the VSVr composition of this invention is interleukin-12.

The VSVr composition is desirably administered in a pharmaceutically acceptable diluent, excipient or vehicle, such as those described below. Although the composition can be administered by any selected route of administration, in one embodiment a desirable administration procedure is intra-
nasal.

In an embodiment of the procedure of this invention in which the composition of VSVr is the composition of stimulus, the procedure includes administration of at least one VSVr immunogenic composition after administration of a Immunogenic DNA priming composition. The composition of VSVr expresses the same antigen that is expressed with the composition of priming. In one embodiment the composition of VSVr includes a virus Additional recombinant encoding a selected cytokine. Yet In another embodiment, the VSVr includes a sequence that expresses a cytokine, for example, IL-12 present therein VSVr when the antigen is expressed. Still in another embodiment it administer multiple VSVr compositions as stimuli later. In one embodiment at least two are administered VSVr compositions after priming compositions. In other embodiment at least three VSVr compositions are administered after Priming compositions.

Desirably, as described above, each subsequent VSVr composition has a different serotype, but the same antigen coding sequence. The different serotypes are selected among serotypes of natural origin and among any synthetic serotype provided by the manipulation of VSV G protein. Among the known methods for altering the VSVr G protein are the technology described in International Publication No. W099 / 32648 and Rose, NF et al. 2000 J. Virol. , 74: 10903-10.

In another embodiment, each VSVr has a different antigen coding sequence, but the same VSV G protein. In yet another embodiment each VSVr has a different antigen coding sequence and a different VSV G protein. As detailed in the examples, one embodiment of a series of VSVr stimulus compositions comprises two optimized VSVr containing an HIV-1 gp160 env gene, with a VSVr which is the Indiana serotype and the other the Chandipura serotype.

In accordance with the present invention this VSVr immunogenic composition can be administered as a stimulus composition subsequently to the administration of the immunogenic composition of priming DNA presenting the same host antigen. In any of the embodiments of the method of the invention is administered the second and any Additional VSVr as a stimulus after the first administration of VSVr. Additional VSVr stimuli in one embodiment are of same serotype that carry the same antigen. Alternatively VSVr stimuli that present different serotypes are administered serially before administration of the composition immunogenic stimulus DNA. It has been shown to be useful Administer at least three stimuli. When used as a Stimulation composition, VSVr compositions are administered in series, following the immunogenic compositions of priming DNA. When used as the priming composition the composition VSVr immunogenic is administered once or preferably several times. Additional VSVr stimuli in one embodiment are of same serotype that carry the same antigen. Alternatively Additional VSVr stimuli that have different serotypes are administered serially before administration of the composition immunogenic stimulus DNA.

Examples of suitable VSVr constructs that are capable of expressing an HIV-1 protein in vivo are described in detail in the following examples, and in the following publications, for example, Rose et al., 2000 Virol. , 268: 112-121; Rose et al., 2000 J. Virol. , 74: 10903-10910; Rose et al. 2001 Cell, 106: 539-549; Rose et al., 2002 J. Virol. , 76: 2730-2738; Rose et al., 2002 J. Virol. , 76: 7506-7517. Additional VSVr vectors can be further attenuated, either by progressive truncates of the VSV G protein, or by randomization of the VSV structural gene as mentioned above. VSV non-replicating can also be used in accordance with this invention. VSVr that exhibit a desired balance of attenuation and immunogenicity is expected to be useful in this invention.

Several illustrative VSVr constructions exemplified in the following examples have genomes recombinants:

(one)

3'NPMG-HIV env -L-5 '

(2)

3'NPMG-HIV gag -L-5 '

(3)

3'NPMG-VIS gag -L-5 '.

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In one embodiment, an immunogenic method and composition of the present invention uses one of these VSVr constructs. In another embodiment, an immunogenic method and composition of the present invention uses both a VSVr-HIV env and a VSVr-HIV gag . This last imungenic composition simultaneously gives rise to potent humoral immune responses for gp160, plus authentically configured CTL (for Env and Gag) capable of killing cells infected with HIV-1 virus. HIV-1 gp160 expressed in this way binds to CD4 / co-receptor and undergoes conformational changes associated with receptor binding. Appropriate gp160 / receptor interactions expose determinants of neutralization of cryptic viruses present in gp160 that are required for the induction of antibody-mediated protection against infection (see, for example, LaCasse, RA et al., 1999 Science . 283: 357- 62).

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C. Antigens for use in immunogenic compositions of this invention

The antigenic or immunogenic compositions useful in the methods and compositions of this invention improve the immune response in a vertebrate host for a selected antigen. The selected antigen, when expressed by plasmid DNA or VSV, it can be a protein, polypeptide, peptide, fragment or a fusion thereof derived of a pathogenic virus, bacteria, fungus or parasite. So alternatively the selected antigen can be a protein, polypeptide, peptide, fragment or fusion thereof derived from a cancer cell or tumor cell. In another embodiment the Selected antigen can be a protein, polypeptide, peptide, fragment or fusion thereof derived from an allergen so that interferes with the production of IgE so that it moderates allergic responses to the allergen. In yet another embodiment the Selected antigen can be a protein, polypeptide, peptide, fragment or fusion thereof derived from a molecule or part of the same that represents those produced by a host (a autocell) in an unwanted way, quantity or location, such as those of the amyloid precursor protein, so that disease characterized by deposition of is prevented or treated Amyloid in a vertebrate host. In an embodiment of this invention, the selected antigen is a protein, polypeptide, HIV-1 derived peptide or fragment.

The invention also relates to procedures. to increase the capacity of an immunogenic composition that contains an antigen selected (1) from a pathogenic virus, bacteria, fungus or parasite to develop the immune response of a vertebrate host, or (2) of a cancer antigen or antigen associated with a cancer cell tumor or tumor cell for develop a therapeutic anti-cancer effect or prophylactic in a vertebrate host, or (3) of an allergen so that interferes with the production of IgE so that it moderates allergic responses to the allergen, or (4) of a molecule or part of the same that represents those produced by a guest (a autocell) in an unwanted way, quantity or location of so that it reduces an unwanted effect.

In another embodiment, immunogenic compositions desirable virals that use the priming / stimulus regime of this invention include those aimed at prevention and / or treatment of disease caused by, without limitation, virus human immunodeficiency, simian immunodeficiency virus, virus respiratory syncytial, parainfluence virus types 1-3, influenza virus, herpes simplex virus, human cytomegalovirus, hepatitis A virus, hepatitis B, hepatitis C virus, human papillomavirus, polio virus, rotavirus, calicivirus, measles virus, mumps virus, rubella virus, adenovirus, virus rabies, canine distemper virus, equine plague virus, human metapneumovirus, avian pneumovirus (formerly virus of the turkey rhinotracheitis), Hendra virus, Nipah virus, coronavirus, parvovirus, infectious rhinotracheitis virus, feline leukemia, feline infectious peritonitis virus, avian infectious bursal disease, disease virus Newcastle, Marek's disease virus, syndrome virus respiratory and reproductive pig, equine arteritis virus and various encephalitis viruses

In another embodiment, desirable bacterial immunogenic compositions using the priming / stimulation regime of this invention include those that are directed to the prevention and / or treatment of diseases caused by, without limitation, Haemophilus influerazae (both typifiable and nontypable ), Haenaophilus somnus, Moraxella catarrhalis, Streptococcus praeunaoniae, pyogefaes Streptococcus, Streptococcus agalactiae, Streptococcus faecalis, Helicobacter pylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bofdetella pertussis, Alloiococcus otiditis, Salmonella typhi, typhirnurimn Salmonella, Salmonella choleraesuis, Escherichia coli, Shigella, Vibrio cholerae, Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium iyatracellulare complex, Proteus nairabilis, Proteus vulgaris, Staphylococcus aureus, Stapliylococcus epidemsaielis, Clostridium pyramids ans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurella multocida, Actinobacillus pleuroprzeumoniae and Mycoplasma gallisepticurn .

In another embodiment, desirable immunogenic compositions against fungal pathogens using the priming / stimulus regime of this invention include those that are directed to the prevention and / or treatment of diseases caused by, without limitation, Aspergillis, Blastomyces, Candida, Coccidiodes, Cfyptococcus and Histoplasnza .

In another embodiment, desirable immunogenic compositions against parasites using the priming / es- regimen regime
The terms of this invention include those that are directed to the prevention and / or treatment of diseases caused by, without limitation, Leishmania major, Ascaris, Trichuris, Giardia, Schistosoma, Cryptosporidium, Trichomonas, Toxoplasma gondii and Pneumocystis carinii.

In another embodiment, immunogenic compositions desirable to give rise to an anti-cancer effect therapeutic or prophylactic in a vertebrate host, who use the priming / stimulus regime of this invention include those that use a cancer antigen or tumor associated antigen including, no limitation, specific antigen, antigen carcino-ambrionic, MUC-1, Her2, Ca-125 and MAGE-3.

Nucleotide and protein sequences for known antigens listed above are found publicly available through databases such as NCBI, or can be found available in other sources such as American Type Culture Collection and universities.

Desirable immunogenic compositions for moderate responses to allergens in a vertebrate host, who use The priming / stimulus regime of this invention includes those that contain an allergen or fragment thereof. Examples of such Allergens are described in U.S. Patent No. 5,830,877 and international patent publication number WO 99/51259. Such allergens include, without limitation, pollen, insect venomas, animal dander, fungal spores and drugs (such as penicillin). These immunogenic compositions interfere with the IGE antibody production, a known cause of reactions allergic

Desirable immunogenic compositions for moderation of responses to own molecules in a host vertebrate, which use the priming / stimulus regime of this invention, include those that contain their own molecule or fragment of it. Examples of such proprietary molecules include the β-chain insulin involved in diabetes, the molecule G17 involved in gastroesophageal reflux disease, and antigens that sub-regulate autoimmune responses in diseases such as multiple sclerosis, lupus and rheumatoid arthritis. I also know includes the β-amyloid peptide (also designated as Aβ peptide), which is a fragment from 39 to 43 amino acids, internal, of amyloid precursor protein (APP) that are generated by APP processing by the enzymes? and γ-secretase. All 42 peptides have the next sequence SEQ ID No. Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala.

It is also desirable in the selection and use of antigenic sequences for design of DNA plasmids and VSVr constructs of this invention alter the use of the codon of the gene sequence encoding the selected antigen, as well as also the plasmids of DNA in which they are inserted, and / or eliminate inhibitory sequences inside. The elimination of inhibitory sequences can be carried out using the technology described in detail in United States patents United numbers 5,965,726; 5,972,596; 6,174,666; 6,291,664; Y 6,414,132; and in international patent publication no. WO01 / 46408. Briefly described, this technology implies the mutation of inhibitor / instability sequences identified in the selected gene, preferably with point mutations multiple.

As a specific embodiment exemplified below, the immunogenic plasmid and VSVr compositions of this invention desirably use one or more sequences optimized to encode HIV-1 antigens, such as gag, pol and nef antigens, or immunogenic fragments. or mergers thereof. The gag and env genes of the chimeric simian-human immunodeficiency virus (VISH) (89.6P) are useful for preparing RSVV, such that protection against infection and mitigation of virus and disease burden can be demonstrated in a model of Rhesus macaque disease. The following examples demonstrate the use of VISH analogs, ie VIS gag and HIV 89.6P env .

D. Promoters useful in the DNA plasmid or immunogenic VSVr constructs

Promoters suitable for use in any of the components of this invention can be readily selected from constitutive promoters, inducible promoters, tissue specific promoters and others. Examples of constitutive promoters that are not activity specific and used in the nucleic acid molecules encoding an antigen of this invention include, without limitation, the retroviral Rous sarcoma virus (VSR) promoter, the retroviral LTR promoter (optionally with the VSR enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, for example, Boshart et al., 1985 Cell, 41: 521-530), the SV40 promoter, the dihydrofolatoreductase promoter, the β-actin promoter, the phosphoglycerolkinase (PGK) promoter and the EF1α promoter (Invitrogen). Inducible promoters that are regulated by exogenously supplied compounds, include, without limitation, the arabinose promoter, the zinc inducible sheep metallothionin (MT) promoter, the dexamethasone inducible murine breast tumor virus (VTMM) promoter (Dex ), the promoter system of T7 polymerase (WO 98/10088); the ecdysone promoter in insects (No et al., 1996 Proc. Natl. Acad. Sci. USA , 93: 3346-3351), the tetracycline-containing system (Gossen et al., 1992 Proc. Natl. Acad. Sci. USA , 89: 5547-5551), the tetracycline inducible system (Gossen et al., 1995 Science , 268: 1766-1769, see also Harvey et al., 1998 Curr. Opira. Chern. Biol., 2: 512- 518), the RU486 inducible system (Wang et al., 1997 Nat. Biotech., 15: 239-243 and Wang et al., 1997 Gene Ther., 4: 432-441) and the rapamycin inducible system (Magari et al., 1997 J. Cliva. Invest., 100: 2865-2872).

Other types of inducible promoters that may be useful in this context are those regulated by a specific physiological state, for example, temperature or acute phase or only in replicating cells. Useful tissue-specific promoters include gene promoters encoding skeletal β-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with greater activities than naturally occurring promoters (see Li et al., 1999 Nat. Biotech. , 17: 241-245). Examples of promoters that are tissue specific to the liver are known (albumin, Miyatake et al. 1997 J. Virol. , 71: 5124-32; hepatitis B virus core promoter, Sandig et al., 1996 Gene Ther ., 3: 1002-9; alpha-fetoprotein (AFP), Arbuthnot et al., 1996 Hum. Gene Ther. , 7: 1503-14), bone (osteocalcin, Stein et al., 1997 Mol. Biol. Rep. , 24: 185-96; bone sialoprotein, Chen et al., 1996 J. Bone Miner. Res. , 11: 654-64), lymphocytes (CD2, Hansal et al., 1988 J. Immuol. , 161: 1063 -8; immunoglobulin heavy chain; T cell receptor α chain), neuronal (neuron specific enolase promoter (NSE), Andersen et al. 1993 Cell. Mol. Neurobiol. , 13: 503-15; gene of the neurofilament light chain, Piccioli et al., 1991 Proc. Natl. Acad. Sci. USA , 88: 5611-5; the neuron specific ngf gene, Piccioli et al., 1995 Neuron , 15: 373- 84); among others. See, for example, International Patent Publication No. WO00 / 55335 for additional lists of known promoters useful in this context.

E. Vehicles, excipients, adjuvants and formulations useful for the immunogenic compositions of this invention

Immunogenic compositions useful in the present invention, either the plasmid DNA compositions or of VSVr, further comprise an immunologically acceptable diluent or a pharmaceutically acceptable carrier, such as sterile water or Isotonic sterile saline. The antigenic compositions are they can also mix with diluents or vehicles in a way conventional. As used in the present invention the expression "pharmaceutically acceptable vehicle" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, agents isotonic and absorption delay, and the like, compatible with administration to humans or other invertebrate hosts. He Appropriate vehicle is evident to those skilled in the art and will depend largely on the route of administration.

Additional components that may be present in the immunogenic compositions of this invention are adjuvants, preservatives, surfactants and stabilizers chemicals, suspending or dispersing agents. Typically optimize stabilizers, adjuvants and preservatives for determine the best formulation for efficacy in the human or animal Diana.

1. Adjuvants

An adjuvant is a substance that improves the immune response when administered together with an immunogen or antigen. A number of cytokines or lymphokines have been shown to they have immune modulating activity, and therefore can be used as adjuvants, including, but not limited to, the interleukins 1-?, 1-?, 2, 4, 5, 6, 7, 8, 10, 12 (see, for example, U.S. Patent No. 5,723,127), 13, 14, 15, 16, 17 and 18 (and their mutant forms), the interferons?,? and γ, macrophage colony stimulating factor of granulocyte (see, for example, U.S. Patent No. 5,078,996 and access number ATCC 39900), stimulating factor of the macrophage colony, colony stimulating factor of granulocytes, GSF, and tumor necrosis factors? and \beta. Other adjuvants useful in this invention include a chemokine, including without limitation, MCP-1, MIP-1?, MIP-1?, And RANTES. Adhesion molecules such as a selectin, by example, L-selectin, P-selectin and E-selectin may also be useful as adjuvants Still other useful adjuvants include, without limitation, a mucin-like molecule, for example, CD34, GlyCAM-1 and MadCAM-1, a member of the integrin family such as LFA-1, VLA-1, Mac-1 and p150.95, a member of the superfamily of immunoglobulin such as PECAM, ICAMs, for example, ICAM-1, ICAM-2 e ICAM-3, CD2 and LFA-3 molecules co-stimulators such as CD40 and CD40L, factors growth factors that include vascular growth factor, factor of nerve growth, fibroblast growth factor, epidermal growth factor, B7.2, PDGF, BL-1, and vascular endothelial growth factor, molecules of receiver including Fas, TNF receiver, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still another adjuvant molecule It includes caspase (ICE). See also patent publications International numbers W098 / 17799 and W099 / 43839.

Suitable adjuvants used to improve a immune responses include, without limitation, MPL? (lipid A of 3-O-deacetylated monophosphoryl; Corixa, Hamilton, MT), which is described in the United States patent No. 4,912,094, which is incorporated into this invention as reference. They are also suitable for use as analog adjuvants of synthetic lipid A or phosphate compounds of aminoalkylglucosamine (AGP), or derivatives or analogs thereof, that are available in Corixa (Hamilton, MT), and that are described in U.S. Patent No. 6,113,918, which is incorporates this invention as a reference. An AGP of this type is 2 - [(R) -3-tetradecanoyloxytetradecanoylamino]  ethyl 2-deoxy-4-0-phosphono-3-0 - [(R) -3-tetradecanoioxytetradecanoyl] -2 - [(R) -3-tetradecanoyloxytetradecanoyl-amino] -b-D-glucopyranoside,  which is also known as 529 (previously known as RC529). This adjuvant 529 is formulated as an aqueous form or as a stable emulsion

Other adjuvants include mineral oil and water emulsions, aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, etc. amphoteric, avridine, L121 / squalene, D-lactide-polylactide / glycoside, pluronic polyols, muramyl dipeptide, murdered Bordetella , saponins, such as Stimulon ™ QS-21 (Antigenics, Framingham, MA.), described in U.S. Patent No. 5,057,540, which is incorporated herein by reference, and particles generated therefrom such as ISCOMS (immunostimulatory complexes), Mycobacterium tuberculosis, bacterial lipopolysaccharides, synthetic polynucleotides such as oligonucleotides containing a CpG motif (U.S. Patent No. 6,207,646, which is incorporated herein by reference), a pertussis toxin (PT), or a heat labile toxin from E. coli (LT), particularly LT-K63, LT-R72 , PT-K9 / G129; see, for example, international patent publications numbers WO 93/13302 and WO 92/19265. Also useful as adjuvants are cholera toxins and mutants thereof, including those described in published international patent application number WO 00/18434 (in which the glutamic acid in the amino acid of position 29 is replaced with another amino acid (other of aspartic acid), preferably a histidine). CT toxins or similar mutants are described in published international patent application number WO 02/098368 (in which the isoleucine in the amino acid of position 16 is replaced by another amino acid, either alone or in combination with the serine replacement in the amino acid of position 68 with another amino acid; and / or in which the valine in the amino acid of position 72 is replaced with another amino acid). Other CT toxins are described in published international patent application number WO 02/098369 (in which the arginine in the amino acid of position 25 is replaced with another amino acid; and / or an amino acid is inserted in the amino acid of position 49 ; and / or two amino acids are inserted into the amino acid of positions 35 and 36).

In an embodiment exemplified below the desired adjuvant is IL-12, which is expressed in a plasmid See, for example, United States patents numbers 5,457,038; 5,648,467; 5,723,127 and 6,168,923. This plasmid IL-12 expression is incorporated into the composition priming containing immunogenic DNA plasmid of the examples. However, it should be noted that this plasmid is could administer to the mammalian or vertebrate host with the composition of VSVr (or express with the VSVr) or alone, between priming and stimulation compositions. In one embodiment the cytokine It can be administered as a protein. In a preferred embodiment  the cytokine is administered as a nucleic acid composition comprising a DNA sequence that encodes the cytokine under the control of regulatory sequences that direct the expression of same in a mammalian cell. In yet another useful embodiment, the plasmid expressing the cytokine is administered with the composition of DNA. In yet another embodiment the cytokine is administered between the administrations of the priming composition and the composition of stimulus. Still in another stage the cytokite is administered with the stage of stimulus In yet another embodiment the cytokine is administered with both priming and stimulation compositions.

When the priming composition is the DNA plasmid composition, as in the following examples, the plasmid composition may comprise a DNA sequence that encodes the cytokine under the control of regulatory sequences which direct their expression in the mammalian cell. In some embodiments the cytokine coding sequence is present in the same DNA plasmid as the sequence that encodes the cytokine. Still in other embodiments the sequence that encodes the antigen. Even in other embodiments, the sequence that encodes the cytokine is present in a different DNA plasmid of the DNA plasmid encoding the antigen.

2. Facilitating agents or co-agents

In addition to a vehicle as described previously, the immunogenic compositions constituted by polynucleotide molecules desirably contain agents optional polynucleotide facilitators or "co-agents", such as a local anesthetic, a peptide, a lipid that includes cationic lipids, a liposome or lipid particle, a polycation such as polylysine, a polycation in three-dimensional branched such as a dendrimer, a carbohydrate, a cationic amphiphile, a detergent, a benzylammonium surfactant, or other compound that facilitates the transfer of polynucleotides to cells. Such facilitating agent includes bupivacaine or anesthetic tetracaine (see patents United States numbers 5,593,972; 5,817,637; 5,380,876; 5,981,505 and 6,383,512 and international patent publication number WO 98/17799). Other non-exclusive examples of such are described. agents or co-facilitators useful in this invention in United States patents numbers 5,703,055; 5,739,118; 5,837,533; International Patent Publication Number No. WO96 / 10038, published April 4, 1996; and publication of international patent number WO94 / 16737, published on August 8 of 1994.

Most preferably, the local anesthetic is present in an amount that forms one or more complexes with the nucleic acid molecules. When the local anesthetic mixes with nucleic acid molecules or plasmids of this invention, this form a variety of small complexes or particles that package DNA and are homogeneous. Therefore, in an embodiment of the Immunogenic compositions of this invention, the complexes are formed by mixing the local anesthetic and at least one plasmid from the present invention Any simple complex that results from this mixture can contain a variety of combinations of Different plasmids Alternatively in another embodiment of the compositions of the present invention, the local anesthetic is You can pre-mix with each plasmid separately. Be then combine the mixtures separately in a single composition to ensure that the desired plasmid ratio is present in a simple immunogenic composition, if all plasmids are they have to administer in a simple bolus administration. From alternatively the local anesthetic and each plasmid can be mix separately and administered separately to obtain the desired relationship

When the term is used hereafter "complex" or "one or more complexes" or "complexes" to define this embodiment of the immunogenic composition, it understands that the term comprises one or more complexes. Every complex contains a mixture of plasmids, or a mixture of discreetly formed complexes. Each complex can contain only a type of plasmid or complex, or a mixture of plasmids or complexes, in which each complex contains a polycistronic DNA. Preferably the complexes are between about 50 and approximately 150 nm in diameter. When the facilitating agent used is a local anesthetic, preferably bupivacaine, it prefers an amount of about 0.1 percent by weight to approximately 1.0 percent by weight based on the total weight of the polynucleotide composition. See also the publication of International Patent Number WO 99/21591, and which shows the incorporation of benzylammonium surfactants such as co-agents, preferably administered in a amount between about 0.001 and 0.03% by weight. In accordance with the present invention the amount of local anesthetic is present in a ratio to said nucleic acid molecules of 0.01 to 2.5% in p / v of local anesthetic to 1 to 10 µg / ml of nucleic acid. Another range of this type is 0.05 to 1.25% w / v anesthetic. local up to 100 µg / ml at 1 mg / ml nucleic acid.

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3. Other additives for the compositions immunogenic

Other additives may be included in the immunogenic compositions of this invention, including preservatives, stabilizing ingredients, surfactants and Similar.

Suitable example preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethylvaniline, glycerin, phenol and parachlorophenol

Suitable stabilizing ingredients that they can use include, for example, casamino acids, sucrose, gelatin, phenol red, N-Z amine, bisphosphate monopotassium, lactose, hydrolyzed lactalbumin and dry milk.

Suitable surfactants include, without limitation, incomplete Freund's adjuvant, quinone analogues, hexadecylamine, octadecylamine, esters of octadecylamine acid, lysolecithin, dimethyl dioctadecylammonium bromide),  methoxyhexadecylglycerol, and pluronic polyols; polyamines, by example, pyran, dextransulfate, poly IC, carbocol; peptides, for example, peptide and dipeptide of muramyl, dimethylglycine, tuftsin; emulsions in oil; and mineral gels, for example, phosphate of aluminum, etc. and immune stimulation complexes (ISCOMS). The Plasmids and VSVr can also be incorporated into liposomes for use as an immunogenic composition. Immunogenic compositions may also contain other additives for the selected mode of Composition administration. The composition of the invention. it may also involve lyophilized polynucleotides, which can be use with other pharmaceutically acceptable excipients for the development of dosage forms of powder, liquid or suspension. See, for example, Remington: The Science and Practice of Pharmacy, vol. 2, 19th edition (1995), for example, chapter 95 Aerosols; and international patent publication number WO99 / 45966.

These immunogenic compositions can contain additives suitable for administration by any conventional administration route. In some embodiments preferred the immunogenic composition of the invention is prepared for administration to human subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, tablets enteric coated or capsules, or suppositories. Therefore the immunogenic compositions may also include, but without limited to these, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes and release formulations Sustained implantable or biodegradable. In an embodiment of a formulation for parenteral administration, the ingredient active is provided in dry form (i.e., powder or granular) for reconstitution with a suitable vehicle (for example, water without sterile pyrogens) before parenteral administration of the reconstituted composition Other formulations administrable by Useful parenteral pathways include those that comprise the active ingredient in microcrystalline form, in a preparation liposomal, or as a component of a polymer system biodegradable. Compositions for sustained release or implant may comprise polymeric or hydrophobic materials pharmaceutically acceptable such as an emulsion, a resin of ion exchange, a slightly soluble polymer or a little salt soluble.

The immunogenic compositions of the present invention are not limited with the selection of vehicles, adjuvants or other physiologically acceptable ingredients conventional, or other ingredients useful in preparations Pharmaceuticals of the types described above. The preparation of these pharmaceutically acceptable compositions, of the components described above, presenting isotonicity at Appropriate pH, stability and other conventional features They are available to specialists in the art.

F. Dosages and administration routes for immunogenic compositions of the present invention

In general, the selection of the appropriate "effective amount" or dosage for the components of the immunogenic composition (s) of the present invention will also be based on the identity of the antigen in the composition (s).
Immunogenic tion (s) used (s), as well as in the physical condition of the subject, most especially including the general health, age, and weight of the immunized subject. The method and routes of administration and the presence of additional components in the immunogenic compositions may also affect the dosages and amounts of the plasmid and VSVr compositions. Such selection and adjustment up or down of the effective dose is within the competence of the person skilled in the art. The amount of plasmid and VSVr required to induce an immune response, preferably a protective response, or produce an exogenous effect on the patient without significant adverse side effects varying depends on these factors. Appropriate doses are readily determined by those skilled in the art.

The antigenic or immunogenic compositions of This invention is administered to a human or non-human vertebrate with a variety of routes that include, but are not limited to, intranasal, oral, vaginal, rectal, parenteral, intradermal, transdermal (see, for example, patent publication International No. WO 98/20734) intramuscular, intraperitoneal, subcutaneous, intravenous and intraarterial. The appropriate route is select based on the nature of the composition used immunogenic, and an evaluation of age, weight, sex and health general of the patient and the antigens present in the composition immunogenic, and similar factors by a physician in charge.

In the examples provided below, Immunogenic DNA compositions are administered via intramuscular (i.m.). In one embodiment it is desirable to administer the VSVr compositions intranasally, rather than im. Without However, the selection of dosages and administration routes does not they are limitations to this invention.

Similarly, the order of administration of immunogenic composition and periods of time between individual administrations can be selected by the doctor in charge or a specialist in the technique based on the physical characteristics and precise responses of the host for the Application of the procedure. It is expected that such optimization will be find in the specialization of the technique.

G. Kit components

In yet another embodiment the present invention provides a pharmaceutical kit for simple administration of a immunogenic, prophylactic or therapeutic regimen for treatment of any of the diseases or conditions above cited for which an immune response to an antigen is desired. He kit is designed for use in an induction procedure of specific immune response of high level antigen in a subject mammal or vertebrate. The kit contains at least one composition immunogenic comprising a DNA plasmid comprising a DNA sequence encoding an antigen under the control of regulatory sequences that direct its expression in a mammalian or vertebrate cell. Preferably provided multiple pre-packaged dosages of the immunogenic composition of DNA in the kit for administrations multiple. The kit also contains at least one composition immunogenic comprising a replication component, virus recombinant vesicular stomatitis (RSVR) comprising a nucleic acid sequence encoding the same antigen under the control of regulatory sequences that direct the expression of same in a mammalian or vertebrate cell. Preferably it provide pre-packaged dosages multiple of the VSVr immunogenic composition in the kit for multiple administrations

While immunogenic compositions described above do not contain plasmids of DNA and / or VSVr that express a cytokine, such as IL-12, the kit it also optionally comprises a cytokine composition separate or multiple pre-packaged dosages of the cytokine composition for multiple administrations. These cytokine compositions are generally compositions of nucleic acid comprising a DNA sequence encoding the cytokine selected under the control of regulatory sequences which direct its expression in a mammalian cell or vertebrate.

The kit also contains instructions for use of the immunogenic compositions in a process of priming / stimulation as described in this invention. The kits can also include instructions for carrying out certain tests, various vehicles, excipients, diluents, adjuvants and the like described above, as well as equipment for administration of the compositions, such as syringes, spray devices, etc. Other components may include disposable gloves, decontamination instructions, applicator bars or containers, among other compositions.

In order to better understand this invention the following examples are described. The examples are For illustrative purposes only and are not intended as limitations of scope of the invention. All documents, publications and patents cited in the following examples are incorporated as reference to this invention.

As demonstrated in the following examples a priming / stimulus protocol of this invention induces in the subject immunized a surprising synergistic effect on cellular responses antigen specific and humoral immune. In fact when these responses induced by a priming / stimulus protocol of This invention is compared with the results of administration of multiple priming compositions alone or stimulus compositions multiple alone, the synergistic nature of the response to Compositions of this invention is drastically evident. The combination of the presentation of the desired antigen by a administration of plasmid DNA followed by a stimulus of VSVr produces an increase in antigen-specific T cells in the immunized subject, who is considerably in excess of any additive response. Similarly the increase shown in the humoral response to the desired antigen is unexpectedly high with the use of the plasmid compositions of DNA and VSVr immunogenic in an immunization protocol. See, for example, Table 1 below and Figures 4 and 5.

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Example 1 Preparation of DNA plasmids

This example describes illustrative plasmids useful in an embodiment of this invention as described in Examples 2 and 3. These plasmids are not a limitation in the present invention but have been optimized for use in subsequent experiments. The following immunogenic DNA compositions were designed using recombinant DNA techniques. The DNA skeleton vector expressing HIV or VIS gag genes uses the CMVH promoter, BGH poly A termination sequence, the bacterial origin of replication CoIEI (hear); and a kanamycin resistance gene for selection.

A. VIS gag p37 DNA plasmid

Plasmid WLV102 is a bacterial plasmid that expresses as the selected antigen against which an immune response was desired, the gag p37 of SIV. Plasmid WLV102 (4383bp) consists of a truncated gag gene optimized with RNA (p37) from VIS (Qiu et al., 1999 J. Virol. , 73: 9145-9152) inserted into the expression vector WLV001 of the DNA plasmid. The gag gene was optimized with RNA by inactivating the inhibitory sequences, thus allowing expression of gag gene independent of high-level Rev, using the technology described in detail in US Pat. Nos. 5,965,726; 5,972,596; 6,174,666; 6,291,664; and 6,414,132; and in international patent publication number WO01 / 46408.

The plasmid skeleton WLV102 comprises three genetic units. The first is an eukaryotic gene expression unit that contains genetic elements of the CMVH immediate early promoter / enhancer (Boshart et al. 1985, cited above) and the BGH polyadenylation signal (Goodwin EC and Rottman FM, 1982 J. Biol Chem. 267 : 16330-16334). The gag gene is cloned between the SalI and EcoRI sites. The second component is a chimeric kanamycin resistance (km '), adenyl 4'-nucleotidyltransferase type Ia gene (see US Patent No. 5,851,804). It has been observed that this gene confers resistance to a limited number of aminoglycosides while allowing the selection of bacteria containing the plasmid. The third component is a CoIEI bacterial origin of replication that is required for propagation of the plasmid during bacterial fermentation. This plasmid is illustrated in Figure 1A.

B. Plasmids encoding a cytokine - DNA from IL-12r

IL-12 WLV104 plasmid from Rhesus is a dual promoter construct that expresses the form rhesus heterodimeric IL-12. Plasmid WLV103 is a dual promoter expression plasmid comprising two genes that encode the proteins of human IL12 p35 and p40. He Plasmid has a total of 6259 nucleotides. Each cistron in WLV103 which contains one of the two interleukin 12 subunits, p35 or p40, is under the control of separate regulatory elements. The p35 subunit is under the control of CMVH promoter / enhancer, and SV40 polyadenylation signal (cloned between SalI sites and MluI). The p40 subunit is under the control of the SCMV promoter and presents a polyadenylation signal (cloned at the site XhoI).

The plasmid skeleton comprises several components. The first is an eukaryotic gene expression unit that contains genetic elements of the CMVH immediate early promoter / enhancer and the SV40 polyadenylation signal (Fitzgerald M and Shenk T., 1981 Cell , 24: 251-260). The second component is a eukaryotic gene expression unit, composed of the CMVS promoter (Jeang et al. 1987 J. Virol. , 61: 1559-1570) and the BGH polyadenylation signal. The third component is a kanamycin resistance gene (km '), adenyl 4'-nucleotidyltransferase type Ia. The fourth component is an origin of bacterial replication of CoIEI that is required for the propagation of the plasmid in bacteria.

Plasmid vectors were analyzed resulting gag of VIS / gag of HIV and IL-12 DNA by restriction enzyme digestion. The ones were transfected plasmids transiently in rhabdosarcoma cells cultured in DMEM medium + 10% fetal bovine serum (FCS) and antibiotics. These cells were then analyzed for proper expression of the viral proteins that use gag specific monoclonal antibody of HIV (ABI) and rhesus polyclonal serum (from infected animals with VIS) for VIS gag in a Western Blot trial. It was detected IL-12 in the supernatant with ELISA kits (R&D Systems) that detect the p70 protein.

Plasmid vectors were expanded in cells Transformed DH10B grown in LB medium supplemented with kanamycin, and were purified using the Qiagen kit according to the manufacturer specifications The DNA was then analyzed by electrophoresis in an agarose gel against a known pattern.

For use in the following experiments each plasmid was formulated at a concentration of 2.5 mg / ml in bupivacaine 0.25% to a total volume of 4.0 cubic centimeters.

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Example 2 Preparation of recombinant VSV vectors A. VSV genomic cDNA cloning

The genetic background for manipulation of VSV genomic cDNA is pVSV-NX1 (Schnell, MJ, et al 1996 J. Virol. , 70: 2318-23). This clone contains a modified form of the cDNA sequence of the VSV Indiana strain (VSVi). Modifications include the addition of two unique restriction endonuclease recognition sites (XhoI and NheI), and added copies of VSV start gene and VSV end gene signals. When foreign genes such as gp160 env of HIV-1_ {89.6p} or gag p55 of VIS or gag of HIV-1 are conveniently inserted between the XhoI and NheI sites, they reside in a position suitable for expression controlled by signals from SVS transcriptional control. Thus the VSV cDNA sequence is flanked by cis-acting DNA sequences required to promote the rescue of live virus replicates. The T7 RNA polymerase promoter directs the transcription of a primary transcript through the viral cDNA. The ribozyme cleaves the primary transcript to form the end of the RNA genome after the T7 RNA polymerase completes the transcription.

Initially VSVri (pVSV-XN1) was modified genomic clone by insertion of the gag gene (HIV Clade B) between the G and L genes to produce plasmid prVSVri- gag. Similarly, a separate VSVri cDNA clone containing the HIV env gene (pVSVri-env genomic clone) was prepared. The gag and env cDNA sequences were prepared for insertion into pVSV-XN1 by amplification of the coding region sequences in plasmid temperates containing the HIV HXBc2 gag gene or the env gene of the HIV 6101 strain. The primers used for PCR amplification contained terminal restriction enzyme cleavage sites suitable for subsequent cloning; the 5 'primer contained an XhoI site and the 3' primer contained an NheI site. The sequences of the amplified coding region were inserted separately into pVSV-XN1 to generate a clone containing gag and a clone containing env .

The env gene inserted into pVSV-XN1 was a modified form encoding HIV env / VSV G fusion protein. It was shown that Env cell surface expression after infection with a VSVr-HIV Env vector is improved if the cytoplasmic tail of Env was replaced with the shorter cytoplasmic tail of VSV G protein using an overlap PCR procedure ( see, for example, Johnson, et al., 1998 cited above; Johnson et al., 1997 , cited above). The vector skeleton of these two plasmids was altered by changing the Indiana G gene to that of Chandipura or New Jersey serotypes according to published techniques. The G gene exchange was achieved with the advantage of a single MluI site in the M gene and the XhoI site that was designed in the VSV cDNA clones.

The sequences encoding the VSV G gene from the Chandipura strain or the New Jersey strain was amplified with PCR using a 5 'primer that extends through the MluI site within the gene M. A 3 'PCR primer containing sequences homologous to the 3 'end of the G gene as well as additional terminal sequences corresponding to the signal of final gene / start gene and the XhoI site. Sequences of G gene coding amplified with these primers to replace the original G Indiana gene after having exerted from the plasmid skeleton with MluI and XhoI.

B. Rescue of VSVr from cDNA clones

The genomic cDNA plasmid or RNA transcribed from a genomic cDNA plasmid was not enough to start the viral replicative cycle after being introduced into a cell. If same, viral genomic RNA was not an active temperate for Translation or replication. Therefore, the recombinant virus must be recover from the various VSV genomic cDNA constructs. Rescue procedures known in the art have done possible to recover the cloned DNA virus. Virus rescue successful required that VSV genomic RNA be present in the cell together with VSV N protein to encapsidate the RNA genomic viral, as well as P and L proteins, which form the RNA viral RNA dependent polymerase necessary for the synthesis of Viral mRNA and genome replication. The production of all these viral components within cultured cells was achieved by plasmid cotransfection for the VSV genomic cDNA more expression plasmids encoding the N, P and L proteins of VSV

All these plasmids were designed for transcription by phage T7 RNA polymerase; accordingly, cells transfected with a recombinant virus vaccine expressing phage polymerase (MVA / T7 or VTF7-3) were also infected. The conventional procedure used for the rescue of VSV is described in Lawson et al. 1995 , cited above; and Schnell et al., 1996 J. Virol. , 70: 2318-23. This procedure was modified to make it more efficient for the rescue of highly attenuated viruses, and also to make the procedure consistent with regulatory agency guidelines. The procedure is briefly described below.

Qualified Vero cells were transfected in 12.5 cm 2 flasks with a genomic clone of VSVr and supported plasmids encoding the N, P and L genes of VSV using a calcium phosphate transfection procedure. At this time the transfection was initiated, enough certified MVA / T7 was added to provide a multiplicity of infection of 2 plaque formation units per cell. Three hours after the start of the transfection, the cells were subjected to a 3-hour heat shock stage to improve the rescue efficiency of various negative strand RNA viruses (Parks, CL et al., 1999 J. Virol., 73 : 3560-6). At 48-72 hours after transfection the cells and culture medium were transferred to a larger flask containing an established monolayer of Vero cells and subsequently incubated one or two days to allow amplification of the rescued virus. The virus cultured after this amplification stage was filtered to remove most of the contaminating MVA / T7 and was used for clonal isolation of a strain of VSVr.

RNA nucleotide sequence was determined viral genomic by a consensus sequencing procedure to analyze viral RNA genomes. Briefly, it was transcribed inversely purified viral RNA to produce cDNA, and then it amplified regions of genome overlap by PCR that use gene specific primers. Products were gel purified PCR, then underwent cycle sequencing using fluorescent dye terminators. The products were purified from sequencing reaction and were analyzed in a sequencer automated

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C. Specific constructions used in examples

To produce the specific recombinant VSV vectors used in the following experiments, recombinant VSVs expressing 89.6 P gp160 of HIV-1 fused with the transmembrane region of the VSV G protein (VSVr HIV-1envG) and VISmac239 gag were mixed p55 (VSVr VISgag) and were used as the experimental immunogenic composition. An RSVr expressing the influenza hemagglutinin protein (RSV fluHA) was used as a control composition. Recombinant VSV vectors were prepared as previously described (Rose et al., 2000 J. Virol. 74: 10903-10) with the gene encoding the desired antigen inserted between the VSV G and L transcription units. The following construction was described in Rose et al., See above.

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1. Plasmid construction

A plasmid containing Chandipura glycoprotein [G (Ch)] (Masters, PS, 1989 Virol. , 171: 285-290) was favorably provided by Dr. Amiya Banerjee, Cleveland Clinic. To construct the VSV vector containing the G (Ch) gene instead of the Indiana glycoprotein gene [G (I)], an XhoI site was first removed from the G (Ch) gene using oligonucleotide-directed mutagenesis with the 5'-CCCC complementary primers
TAGTGGGATCTCCAGTGATATTTGGAC (SEQ ID NO: 2) and 5'-GTCCAAATATCACTGGAGATCCCACTAGGGG (SEQ ID NO: 3) and the Stratagene QuikChange mutagenesis kit. The mutation of CTCGAG (SEQ ID NO: 4) in CTC C AG (SEQ ID NO: 5) removed the XhoI site without affecting the amino acid sequence of the G (Ch) protein. The gene was then amplified by PCR using Vent DNA polymerase (New England Biolabs). The front primer was 5'-GATCGATCGAATTC ACGCGT AACATGACTTCTTCAG (SEQ ID NO: 6), which contains an Mlu1 (underlined) site upstream of the ATG start codon for protein G (Ch). The reverse primer was 5'-GAACG GTCGAC GCGC
CTCGAG CG TGATATCTGTT AG TTTTTTTCATA TCATGTTGTTGGGCTTGAAGATC (SEQ ID NO: 7) and contained SalI and XhoI sites (bold), followed by VSV transcription start and stop signals (underlined), followed by the complement of the 3 'coding sequence of G (Ch).

The PCR product was digested with MluI and SalI and cloned into the pVSVXN-1 vector (Schnell, et al 1996 J. Virol. 70: 2318-2323) that had been digested with MluI and XhoI to eliminate the G coding sequence (I) of VSV (SalI and ZhoI leave compatible ends for ligation). The plasmid derived from this procedure was designated pVSV (GCh) XN-1 and contains an expression site for foreign genes flanked by unique XhoI and NheI sites between the G (Ch) gene and the L gene.

A procedure identical to that described above was used to generate the vector containing the G (NJ) protein gene of plasmid pNJG (Gallione, CJ, and JK Rose. 1983 J. Virol .: 162-169). The front primer was 5'-GATC
GATCGAATTC ACGCGT AATATGTTGTCTTATCTAATCTTTGC (SEQ ID NO: 8), and the reverse primer was 5'-GGAACG GTCGA CGCGCCTCGAGCG TGATATCTGTT AGT TTTTTTCATA TTAACGGAAATGAGCCATTTCC
ACG SEQ ID NO: 9). The sites indicated by the bold letters and the underlined sequences are as described for the previous Chandipura construct, and the subsequent cloning steps were also described above. The derived final vector plasmid was designated pVSV (GNJ) XN-1 and contains an expression site for foreign genes flanked by unique XhoI and NheI sites between the G (NJ) gene and the VSV L gene.

To generate vectors containing the HIV Env 89.6 G gene, the gene encoding envelope protein 89.6 with the VSV cytoplasmic G tail was cleaved with XhoI and NheI from pVSV-89.6gpl60G (Johnson et al 1997, cited above) and was cloned between the XhoI and NheI sites in pVSV (GNJ) XN-1 or pVSV (GCh) XN-1 vector. This gp160G gene encodes all domains of gp 120 and ecto and transmembrane of gp41 and has four amino acids of the cytoplasmic domain of 89.6 Env (NRVR) (SEQ ID NO: 10) fused with the 26 amino acids of terminal C of the cytoplasmic domain G of VSV , which begins with the IHLC sequence (SEQ ID NO: 11).

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2. Recombinant virus recoveries

Recombinant VSVs were recovered using established procedures (Lawson et al 1995 cited above). Briefly, hamster breeding kidney (BHK) cells were grown to approximately 60% confluence in 10 cm discs. The cells were then infected with a multiplicity of infection (MOI) of 10 with vTF7-3, a recombinant virus vaccine expressing T7 RNA polymerase (Fuerst et al 1987 Mol. Cell. Biol. 7: 2538-2544). After 1 hour each cell disc was transfected with 3 µg of pBS-N, 5 µg of pBS-P, 1 µg of pBS-L, and 10 µg of the plasmid encoding one of the three recombinants in length complete described above. Transfections were carried out with a cationic liposome reagent containing dimethyldioctadecylammonium bromide and dioleyl phosphatidylethanolamine (Rose et al., 1991 Biotechniques 10: 520-525). Cells were then incubated at 37 ° C for 48 hours. Cell supernatants were passed through a 0.2 µm filter to remove most of the virus vaccine and then applied to fresh BHK cells for an additional 48 hours at 37 ° C. For some recoveries an additional plasmid encoding G (pBS-G) of VSV, 4 µg / plate was included with the support plasmids N, P, and L. Infectious virus recovery was confirmed by scanning cell monolayers. BHK for cytopathic effect of VSV. Virus plates were then isolated in BHK cells and virus stock solutions were grown from individual plates by virus addition in a single plate to a 10 cm diameter plate of BHK cells. These stock solutions were then stored at 80 ° C. Titres obtained for VSV (GI) -89.6G, VSV (GCh) -89.6G, and VSV (GNJ) -89.6G were all in the range of 10 7 to 10 8 PFU / ml after freezing and thawing, a procedure that reduces VSV titres approximately three times.

For use in the following experiments each VSVr construction was diluted with a final volume of 0.8 cubic centimeters with sterile DME.

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Example 3 Immunization regime with primer / stimulus A. Immunization protocols

Rhesus macaques were immunized (5 per group) by intramuscular injection with 5 mg of a DNA plasmid bicistronic encoding IL-12 p35 e Rhesus IL-12 p40 in combination with 5 mg of a DNA plasmid expressing VIS gag p37 polyprotein (groups 1 and 2), or 10 mg of an empty DNA plasmid (groups 3 and 4). All these DNA plasmids and formulations thereof are described. in detail in example 1. The immunization schedule with DNA attempted an initial immunization on day 0, followed by a first and second stimulation immunization at week 4 and 8. It they prepared injections at four sites in the deltoids and quadriceps with 1 cubic centimeter per site using a needle and syringe.

The macaques were then reinforced at week 15 by intranasal inoculation (0.4 cubic centimeters / hole with manual pipette) with recombinant vesicular stomatitis virus (VSVr) based on Indiana serotype vector (I) of Example 2 containing gp160env HIV-1 gene (5 x 10 6 pfu) and a second VSVr (I) containing VIS gag p55 gene (5 x 10 6 pfu) (groups 2 and 3), or VSVr (I ) containing influenza hemagglutinin gene (1 x 10 7 pfu) (groups 1 and 4). The macaques were reinforced again at week 23 by intranasal inoculation (0.4 cubic centimeters / hole with manual pipette) with the VSVr-based vector (Chandipuri serotype, Ch) of Example 2 containing HIV-1 gp160 env gene ( 5 x 10 6 pfu) and a second VSVr (Ch) containing VIS gag p55 gene (5 x 10 6 pfu) (groups 2 and 3), or VSVr (Ch) containing hemagglutinin gene from the flu (1 x 10 7 pfu) (groups 1 and 4).

Macaques were closely monitored for induction of cellular and humoral immune responses in blood peripheral and for antibody responses on various surfaces mucosal The follow-up involved the development of the trial enzyme ligand immunospot (ELISpot) for VIS gag p55 peptide for interferon gamma, an ELISpot for peptide pool env 6101 from HIV-1 for interferon gamma and an ELISpot for pool of VSV N peptide for interferon gamma. ELISpot's essay detected cellular immune responses in the PBL.

The humoral immune responses are examined by serum evaluation (figure 4), nasal lavage, secretions rectal and saliva (data not shown) for gag p27 IgG titres anti-VIS by ELISA and titles anti-HIV-lgpl60 env by ELISA (data not revealed). For serum the antibodies used in the ELISAs were normal antibodies to the SHIV89.6 chimeric viruses and SHIV89.

B. ELISpot assay to detect murine cells and human secreting cytokines

The filter immunoplate assay, called also otherwise the enzyme linked immunospot assay (ELISpot) was initially developed to detect and quantify B cells that secrete individual antibodies. The technique originally provided a fast and versatile alternative to Cellular assays by conventional plaque formation. The Recent modifications have improved the sensitivity of the assay ELISpot such that cells that produce about 100 can be detected specific protein molecules per second. These essays take advantage with the relatively high concentration of a given protein (such as a cytokine) in the surrounding environment immediately the cell that secretes protein. These products Cells are captured and detected using high antibody affinity.

The ELISpot assay uses two antibodies high affinity cytokine specific directed against different epitopes in the same cytokine molecule: well two monoclonal antibodies or a combination of an antibody monoclonal and a polyvalent antiserum. ELISpot generates spots based on a colorimetric reaction that detects the cytokine secreted by a single cell. The stain represents a "footprint" of the cell that produces the original cytokine. The spots (i.e. cells that form spots or CFS) are permanent and can be quantified visually, microscopically or electronically

The ELISpot assay was carried out as follows: Ninety-six-well flat bottom ELISpot plates (Millipore, Bedford, MA) were coated overnight with an anti-human interferon? (HIFN-?) Monoclonal antibody mouse (clone 27, BD-Pharmingen, San Diego CA) at a concentration of 1 µg / ml, washed ten times with 1 x PBS supplemented with 0.25% Tween-20 and blocked for 2 hours with PBS containing 5% fetal bovine serum (FBS). Peripheral blood lymphocytes (PBL) from Rhesus macaque were isolated in freshly thawed heparinized whole blood by centrifugation with Ficoll-Hypaque density gradient and resuspended in complete culture medium (RPMI 1640 medium supplemented with 5% FCS, L-glutamine 2 mM, 100 units / ml penicillin, 100 µg / ml streptomycin sulfate, 1 mM sodium pyruvate, 1 mM HEPES, 100 µM non-essential amino acids) containing 50 µg / ml PHA-M (Sigma), a pool of peptides of 15 amino acids overlapping with 11 amino acids that extend to the entire open gag reading frame of VIS (final concentration of 1 µM peptide), or medium
alone.

The numbers of incorporated cells were 2 x 10 5 PBLs in 100 µl / well and analyzed in duplicate wells. Cells were incubated for 16 hours at 37 ° C and then removed from the plate by first washing with deionized water and then 10 times with 1 x PBS containing 0.25% Tween-20. After this the plates were treated with a polyclonal biotinylated anti-hIFN-γ detector antibody (0.2 µg / well, Biosource, Camarillo, CA) diluted with 1 x PBS containing 1% BSA and incubated at temperature atmosphere for 2 hours. The plates were then washed 10 times with 1 x PBS containing 0.25% Tween-20 and treated with 100 µl per well of alkaline streptavidin phosphatase conjugate (Southern Biotech, Birmingham AL) diluted 1: 500 with 1 x PBS containing 5% FBS and 0.005% Tween-20 and incubated for an additional 2.5 hours at room temperature. Unbound conjugate was removed by washing the plate 10 times with 1 x PBS containing 0.25% Tween-20. The chromogenic substrate (100 µL / well, 1-stage NBT / BCIP, Pierce, Rockford, IL) was then added for 3-5 minutes, rinsed with water, the plate was dried with air and the resulting spots were counted eye using a dissecting microscope
inverted

The development of the ELISpot trial of this invention measured the number of CD8 + T cells (CTL) and CD4 + T cells induced in response to the immunization procedure of priming / stimulation of this invention, as measured with production of interferon gamma. This evaluation was carried out in the week 25 for previously treated macaques (after 3 primers of DNA and 2 VSV stimuli).

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C. Results

After immunization with primary DNA, it easily detected ELISpot responses from IF-specific gamma of VIS gag in 8 of 10 macaques immunized with gag / IL-12r DNA from VIS (average 254 SFC / 10 6 cells). After the second immunization with gag / IL-12 DNA from VIS, 10 of the 10 immunized macaques developed great ELISpot responses level (mean 1133 SFC / 10 6 cells) and a third dose of DNA de gag / IL-12 stimulated ELISpot responses gag-specific in a majority of animals (average 1506 cells SFC / 106). After the first stimulus with RSVV HIVenv / VSVr VISgag, ELISpot's gag responses from VIS averages were substantially higher (3772 SFC / 10 6 cells) than the responses that received only one immunization of HIVenv VSVr / simple VISgag VSVr (average 386 SFC / 10 6 cells). These results support the use of an enhanced cytokine DNA primer to increase the immunization of immunization of VSVr.

These results are indicated in Table 1 below and in Figures 2, 3, 4 and 5. Figure 2 shows the N-specific IFN-? ELISpot responses of VSVr in unfractionated peripheral blood mononuclear cells (PBMC) of these animals immunized after week 25. Figure 3 shows ELISpot responses to HIVenv 6101-specific IFN-γ for the same samples. Asterisks indicate where significant differences occurred at p = 0.0001. Figure 4 shows the results of serum antibody responses that measure IgG gag p27 anti-VIS titers by ELISA and gp 160 env titres anti-HIV-1 by ELISA. An immunization protocol with the DNA plasmid encoding the VIS gag protein is depicted with a stimulation of a VSV vector expressing flu HA protein with (black). A protocol of the invention is presented that involves an immunization with plasmid of priming gag DNA followed by a VSV stimulus that expresses HIV gag and env proteins with (blacksquare). A protocol is represented that involves priming immunization with an empty control DNA followed by immunization with a VSV that expresses HIV gag and env proteins with (115 {115}). A protocol is depicted that involves priming immunization with control DNA plasmid followed by immunization with a VSV expressing the flu HA protein with (?). Each group represents results from 5 animals. Statistically significant differences between groups are shown as p = 0.0073 (*); p = 0.5941 (#) op = 0.0027 ( 100 ).

Figure 5 shows the ELISpot responses from SIV gag-specific IFN-? For the same samples. An immunization protocol is represented with the DNA plasmid encoding the VIS gag protein with a stimulus of a VSV vector expressing flu HA protein with (\ blacklozenge). A protocol of the invention is presented which involves an immunization with plasmid DNA priming gag followed of a VSV stimulus that expresses HIV gag and env proteins with (\ blacksquare). It represents a protocol that implies a priming immunization with an empty control DNA followed by immunization with a VSV that expresses HIV gag and env proteins with (\ ding {115}). The (?) Represents a protocol that involves priming immunization with control DNA plasmid followed by immunization with VSV expressing flu HA protein. Every group represents results of 5 animals. Differences are shown statistically significant between groups with parentheses for p = 0.0001 and p = 0.0002.

Table 1 indicates the same results in form tabular.

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TABLE 1 ELISpot responses to IFN- \ gamma specific of VIS gag in rhesus macaque after priming with IL-12 gag DNA from VIS / rhesus and a stimulus with VSVr-VISgag / VSVr-HIVenv

one

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The results of these tests demonstrate a surprising synergistic effect of the priming / stimulus regime according to this invention when compared with the results of administration of single priming compositions and multiple single stimulus compositions. The combination of the presentation of the desired antigen with an administration of plasmid DNA followed by a stimulus of RSVV causes an increase in antigen-specific T cells in the immunized subject, that is, considerably above any additive response. Similarly, the demonstrated increase in humoral response to the desired antigen is unexpectedly high with the use of both the DNA plasmid and the VSVr immunogenic compositions in a protocol of
immunization.

Example 4 Macaque model for HIV immunization

Rhhesus macaques (Rh) were immunized using the priming / stimulation strategy of Example 3 and the VSV plasmids and vectors described in this invention according to the same protocols. Approximately 32 weeks after the first administration of priming composition, the macamos were exposed to a dose of 330 infectious doses of 50% monkey (MID50) of recombinant pathogenic VIS / HIV virus VISH89.6P (Reimann et al. ., 1996 J. Virol. , 70: 3198-3206; Reimann et al 1996 J. Virol. , 70: 6922-6928).

Approximately 50 to 70 days after exposure Animals are controlled in terms of disease. Be control animals for induction of responses mediated by cells and antibody immediately before, and one and two weeks after each immunization. The collected serum is tested before exposure and 2, 4, 6, 8 and 12 weeks after exposure for neutralization of antibody responses against HIV-1 89.6, 89.6P, 6101 and other primary isolates Clade B. During the immunization phase of the experiment, immediately before and every two weeks after exposure, it controlled CD4 / CD8 counts. Immediately before and every week after exposure, viral loads are determined in serum by branched DNA analysis. Other fluids are examined body (vaginal, rectal and nasal secretions) of the animals, as well as saliva) in relation to immune responses Cellular and humoral.

Immune responses mediated by cell for the desired antigen using several of the assays based  in more appropriate cells, which include the CTL release assay of 51 Cr, soluble MHC class I tretamer staining, assay by ELISpot, and intracellular cytokine analysis. They were evaluated mucosal antibody responses by ELISA techniques optimized for use with mucosal samples. Responses to serum antibody by conventional ELISA techniques. further serum from all immunized animals is examined for responses of neutralization antibody against env 6101 of HIV and others Clade B primary isolates. Figure 6 illustrates that the answers high immune levels facilitated by combinations of primer / stimulus of this invention lead to greater protection against AIDS, measured by a reduced loss of CD4 T cells for at least 250 days after exposure.

In addition to controlling immune responses facilitated by the immunogenic composition, the potential is assessed of transmission of the live virus vector by determining the level and duration for which you move. Nasal washes were tested obtained at frequent intervals during the first three weeks after each immunization with respect to the presence of VSV alive. The plasma viral load is also examined for presence of copies of live virus. Figure 7 shows that higher immune responses facilitated by combinations of priming / stimulation results in a reduction in circulating virus in plasma for at least 250 days after exposure.

The results of such exams are also probably high content of antigen specific T cells CD8 + and CD4 + in animals immunized according to this invention in contrast to control animals. It is concluded that the immunized animals according to the methodology of priming / stimulation of this invention will maintain health after HIV exposure while non-immunized animals They will develop AIDS.

It is concluded that a comparison of results of this protocol with other methodologies of known priming / stimulus demonstrates that the procedure of the The present invention has safety advantages for animals immunized and in facilitating higher levels of CTL anti-HIV and antibodies provided by immunization with one or multiple immunizations with composition of primed with DNA alone or with one or multiple immunizations with VSVr vector alone. Repeated priming / stimulation according to this invention is probably synergistic and therefore prophylactically beneficial for subjects pre-exposed and therapeutically beneficial for subjects already infected with HIV.

It is expected that various modifications and minor alterations in the procedure and components will be evident to those skilled in the art.

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<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 31

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Complementary primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

\ hskip1cm 4

         \ vskip0.400000 \ baselineskip
      

<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 31

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Complementary primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

\ hskip1cm 5

         \ vskip0.400000 \ baselineskip
      

<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 6

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Complementary primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

\ hskip1cm 6

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 6

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Complementary primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

\ hskip1cm 7

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 36

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Complementary primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

\ hskip1cm 8

         \ vskip0.400000 \ baselineskip
      

<210> 7

         \ vskip0.400000 \ baselineskip
      

<211> 70

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Reverse primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 7

\ hskip1cm 9

         \ vskip0.400000 \ baselineskip
      

<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 46

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Front primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 8

\ hskip1cm 10

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 73

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Reverse primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

\ hskip1cm eleven

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 4

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Env 89.6 G HIV gene

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

\ hskip1cm 12

         \ vskip0.400000 \ baselineskip
      

<210> 11

         \ vskip0.400000 \ baselineskip
      

<211> 4

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> G cytoplasmic tail of VSV

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 11

\ hskip1cm 13

Claims (24)

1. Use of a first composition comprising a DNA plasmid comprising a DNA sequence that encodes an antigen under the control of regulatory sequences that direct the expression thereof by said plasmid DNA in the preparation of a medicine to induce an immune response specific for the antigen in a mammalian subject, in which the induction of said antigen specific immune response understands

(to)

administer to said subject a effective amount of said first composition; Y

(b)

administer to said subject a effective amount of a second composition comprising a virus of recombinant vesicular stomatitis (VSV) comprising a nucleic acid sequence encoding said antigen under the control of regulatory sequences that direct its expression by said recombinant VSV.

         \ vskip1.000000 \ baselineskip
      

2. Use of a second composition comprising a recombinant vesicular stomatitis virus (VSV) that comprises a nucleic acid sequence encoding said antigen under the control of regulatory sequences that direct the expression thereof by said recombinant VSV in the preparing a medication to induce a response antigen-specific immune system in a mammalian subject, in which induction of said antigen specific immune response understands

(to)

administer to said subject a effective amount of a first composition comprising a plasmid of DNA comprising a DNA sequence encoding an antigen under the control of regulatory sequences that direct expression thereof by said plasmid DNA; Y

(b)

administer to said subject a effective amount of said second composition.

         \ vskip1.000000 \ baselineskip
      

3. Use of

(to)

a first composition comprising a DNA plasmid comprising a DNA sequence that encodes an antigen under the control of regulatory sequences that direct its expression by said DNA plasmid; Y

(b)

a second composition comprising a stomatitis virus recombinant vesicular (VSV) comprising an acid sequence nuclei encoding said antigen under sequence control regulators that direct its expression through said VSV recombinant in the preparation of a medicament to induce a antigen specific immune response in a subject mammal.

         \ vskip1.000000 \ baselineskip
      

4. Use in accordance with any one of the claims 1 to 3, wherein said VSV is competent to replication or non-replicant. 5. Use in accordance with any one of the claims 1 to 3, wherein said first composition is administer to said subject at least once before administration of said second composition. 6. Use according to claim 5, in which said second composition is administered to said subject at less once. 7. Use in accordance with any one of the claims 1 to 3, wherein said second composition is administer to said subject at least once before administration of said first composition. 8. Use according to claim 7, in the that said first composition is administered to said subject at least one time. 9. Use in accordance with any one of the claims 1 to 3, wherein said medicament comprises also a cytokine and in which (a) or (b) further comprises the administration of an effective amount of said cytokine to said mammal. 10. Use according to claim 9, in which said cytokine that is administered in (a) is a composition of nucleic acid comprising a DNA plasmid comprising a DNA sequence encoding a cytokine under the control of regulatory sequences that direct its expression by said DNA plasmid. 11. Use according to claim 9, in which said cytokine that is administered in (b) is a protein. 12. Use according to claim 10 or the claim 11, wherein said cytokine is selected from group consisting of IL-12, IL-15, GM-CSF and a combination thereof. 13. Use according to claim 10, in which said sequence encoding the cytokine is present in the same DNA plasmid as said sequence encoding the antigen or is present in a DNA plasmid different from said plasmid DNA encoding said antigen. 14. Use in accordance with any one of the claims 1 to 3, wherein said antigen is a protein, polypeptide, peptide, a fragment or a fusion thereof, in the that said protein is derived from a member selected from the group consisting of a bacterium, a virus, a fungus, a parasite, a cancer cell, an allergen and a car molecule. 15. Use according to claim 14, wherein said antigen is a human or ape immunodeficiency virus antigen selected from the group consisting of gag, pol, env, nef, vpr, vpu, vif and tat and fragments immunogenic or fusions thereof. 16. Use in accordance with any one of the claims 1 to 3, wherein said first composition comprises (i) a DNA plasmid comprising a DNA sequence which encodes more than one equal or different copy of said antigen or (ii) more than one DNA plasmid, in which each DNA plasmid encodes the same antigen or a different antigen. 17. Use in accordance with any one of the claims 1 to 3, wherein said medicament comprises in addition at least two of said recombinant VSVs and in which (b) it also includes administering at least two of said VSVs recombinant 18. Use according to claim 17, in which each recombinant VSV mentioned presents (i) a protein VSV G different and VSV serotype different, but the same sequence encoding the antigen, (ii) a sequence of different antigen coding, but the same G protein of VSV, or (iii) a different antigen coding sequence and a different VSV G protein. 19. Use in accordance with any one of the claims 1 to 3, wherein said second composition it comprises at least two of said VSVr, in which each VSVr carries a G gene of a different VSV serotype. 20. Use in accordance with any one of the claims 1 to 3, wherein the administration of said second composition comprises serial administration of at least two compositions of VSVr. 21. A kit to induce a response antigen specific immune in a mammalian subject that understands

at least one first composition comprising a DNA plasmid comprising a DNA sequence that encodes an antigen under the control of regulatory sequences that direct its expression by said DNA plasmid; Y

at least one second composition comprising a vesicular stomatitis virus Recombinant (VSV) comprising a nucleic acid sequence encoding said antigen under sequence control regulators that direct its expression through said VSV recombinant

         \ vskip1.000000 \ baselineskip
      

22. The kit according to claim 21, in which said VSV is competent for replication or not replicant 23. The kit according to claim 21, which further comprises a cytokine composition. 24. The kit according to claim 23, wherein said cytokine composition comprises a composition of nucleic acids comprising a DNA plasmid comprising a DNA sequence encoding said cytokine under control of regulatory sequences that direct its expression by said plasmid DNA.